Chelating platform for delivery of radionuclides

ABSTRACT

Siderocalin-metal chelator combinations that bind metallic radioisotopes used in nuclear medicine with high affinity are described. The high affinity siderocalin-metal chelator combinations include a number of chelator backbone arrangements with functional groups that coordinate with metals. The siderocalin-metal chelator combinations can be used to deliver radionuclides for imaging and therapeutic purposes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application based on U.S. patentapplication Ser. No. 16/329,178, filed on Feb. 27, 2019, which is anational phase application based on International Patent Application No.PCT/US2017/048954, filed on Aug. 28, 2017, which claims the benefit ofU.S. Provisional Patent Application No. 62/380,885, filed on Aug. 29,2016, U.S. Provisional Patent Application No. 62/401,687, filed on Sep.29, 2016, and U.S. Provisional Patent Application No. 62/505,458, filedon May 12, 2017, the entire contents each of which are incorporatedherein by reference in their entirety as if fully set forth herein.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under AI094419 awardedby the National Institutes of Health and DE-AC02-05CH11231 awarded bythe Department of Energy. The government has certain rights in theinvention.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is “F053-0053PCT Sequence Listing_ST25.txt”. Thetext file is 76.1 KB, was created on or about Aug. 14, 2016, and isbeing submitted electronically via EFS-Web.

FIELD OF THE DISCLOSURE

The present disclosure provides siderocalin-metal chelator combinationsthat bind metallic radioisotopes used in nuclear medicine with highaffinity. The high affinity siderocalin-metal chelator combinationsinclude a number of chelator backbone arrangements with functionalgroups that coordinate with metals. The siderocalin-metal chelatorcombinations can be used to deliver radionuclides for imaging andtherapeutic purposes.

BACKGROUND OF THE DISCLOSURE

Nuclear medicine refers to the diagnosis and/or treatment of conditionsby administering radioactive isotopes (radioisotopes or radionuclides)to a subject. For example, nuclear medicine can be used to diagnosevarious conditions through imaging, such as positron emission topography(PET) imaging. Therapeutic nuclear medicine is often referred to asradiation therapy or radioimmunotherapy (RIT). Examples of conditionsthat are treated with RIT include various cancers, thyroid diseases,blood disorders, and restenosis following balloon angioplasty and/orstent placement.

Many conditions treated with RIT are associated with uncontrolled orunwanted cell division. When dividing cells are exposed to sufficientlyhigh doses of ionizing radiation, such as in the form of alphaparticles, beta particles, or x-ray or gamma radiation, DNA strandsbreak disrupting the normal process of cell division and inhibiting oreven stopping cellular growth.

While nuclear medicine offers many effective diagnostic and therapeuticuses, there are drawbacks associated with its use. One drawback is thatsites of the body other than the area of diagnostic or therapeuticinterest are affected by the radioactivity, often leading to unwantedside effects. This drawback is caused by release of radioactivity beforearrival at a site of interest.

Attempts have been made to lessen the release of radioactivity beforearrival at a physiological site of interest. In one approach,radionuclides have been attached to chelating agents that are thenattached to a targeting ligand that specifically recognizes and attachesto particular cell types. A common example of such a targeting ligand isan antibody. For example, antibodies targeted to tumor-specific cellsurface markers or other disease-related markers have been chemicallyconjugated to potent synthetic chelating agents such as DOTA(1,4,7,10-tetra-azacylcododecane-N,N′,N″,N′″-tetraacetic acid) and DTPA(diethylenetriamine pentaacetic acid). These chelating agents have thenbeen charged with radioisotopes of the rare earth elements such as Y³⁺or Lu³⁺ or similar trivalent metal ions, such as In³⁺ or Bi³⁺. At leasttwo such radionuclide-conjugated antibodies directed against thetumor-specific cell surface marker, CD20, have been approved for use inhuman patients: Zevalin® (RIT Oncology, LLC, Seattle, Wash.) and Bexxar®(GlaxoSmithKline, LLC, Wilmington, Del.).

U.S. Patent Publication No. 2011/0262353 (Skerra) describes drawbacks ofthe use of antibody-chelator combinations to deliver radionuclides.Skerra notes long circulation times, leading to reduced site-specificdelivery and lowered therapeutic effectiveness and contrast for imaging.Skerra suggests that a solution to this perceived problem would be tocouple targeting ligands that are smaller than antibodies to proteinsthat bind to metal chelators, such as siderocalin (Scn). Scn, also knownas Lipocalin-2 or neutrophil gelatinase-associated lipocalin (NGAL), isa member of the lipocalin family of proteins that binds siderophores, atype of small chelator, with very high affinity (in the sub-nanomolarrange).

Skerra particularly teaches modifying natural Scn to form Scn “muteins”that bind non-natural ligands. Skerra defines non-natural ligands as anycompound which does not bind to native, mature hNGAL under physiologicalconditions. See, for example, Skerra, paragraph [0021]. Thus,non-natural ligands exclude many, if not all, metal and chelated metalcomplexes. More particularly, following alteration of the natural Scnprotein, the Scn muteins bind non-natural ligands that Scn does not bindwith under normal physiological conditions. The alterations to Scninclude mutations at one or more of positions 33, 36, 41, 52, 54, 68,70, 79, 81, 134, 136 and 138. While Skerra's approach increases Scn'sability to bind to targeting ligands that are non-natural Scn bindingpartners, this approach generates other issues with the targeteddelivery of radionuclides described more fully below. Further, even withtargeted delivery, the stability of the radioactive complex (chelatorand radionuclide) is often not strong enough to sufficiently preventearly release of metallic radioisotopes in unintended areas of the body,creating off-target side effects.

There are other challenges associated with the use of nuclear medicine.For example, one beneficial use of nuclear medicine would be toadminister a targeted radioactive imaging complex to ensure that theradioactive complex selectively reaches the physiological site ofinterest. Once selective delivery is confirmed with the imaging complex,a radioactive therapeutic complex could be administered with confidencein its selective and targeted delivery to the site of interest. Inpractice, however, such targeted delivery of a therapeutic cannot beconfirmed with pre-imaging. This is because of two reasons. First,different radionuclides are used for imaging and therapeutic purposesand there are currently no “universal chelators” that can bind all typesof metallic radionuclides. When different chelators must be used for animaging radionuclide versus a therapeutic radionuclide, the activity ofone within the physiological environment is not sufficiently predictiveof the activity of the second. Second, currently available chelators donot effectively shield radionuclides from the physiological environment.This means that radionuclides interact with the physiologicalenvironment following administration and en route to a site of interest.As a result, radionuclides with different charge states (²⁺, ³⁺, ⁴⁺)interact with the physiological environment differently, potentiallyaffecting intended delivery.

Finally, there are significant challenges associated with the efficientmanufacturing and use of ionizing radiation in therapeutic and imagingenvironments.

SUMMARY OF THE DISCLOSURE

The present disclosure provides siderocalin (Scn)-metal chelatorcombinations that bind metallic radioisotopes used in nuclear medicine(e.g., transition metals, f-elements) with high affinity and effectivelyshields the metallic radioisotopes from the physiological environment.The high affinity siderocalin-metal chelator combinations include anumber of chelator backbone arrangements with functional groups thatcoordinate with metals. The siderocalin-metal chelator combinations canbe used to deliver radionuclides for imaging and therapeutic purposes.These disclosed chelating platforms provide numerous benefits.

First, like Skerra, the disclosed chelating platforms utilize Scn.However, the current disclosure teaches that the muteins described inSkerra, designed to increase binding to non-natural ligands (e.g.,targeting ligands) have reduced chelating efficacy. Thus, Scn utilizedin the currently disclosed Scn-metal chelator combinations do notinclude mutations that reduce chelating efficacy. If Scn mutations areused, the mutations maintain or increase, rather than decrease, Scn'schelating efficacy.

Second, the Scn-metal chelator combinations disclosed herein have highaffinity, both between the Scn and metal chelator and between the metalchelator and radionuclide. The high affinity between each of thesecomponents reduces early release of radioactivity, reducing side effectsassociated with the use of nuclear medicine.

Third, the current disclosure provides a universal chelating platformthat accommodates metallic radioisotopes used in nuclear medicine andeffectively shields them from the physiological environment followingadministration. By accommodating and effectively shielding metallicradioisotopes used in nuclear medicine, the Scn-metal chelatorcombinations (sometimes referred to as SCCs herein) can be used toadminister a targeted radioactive imaging complex to ensure that theradioactive complex selectively reaches the physiological site ofinterest. Once selective delivery is confirmed with the imaging complex,a radioactive therapeutic complex can be administered, with confidencethat the radioactive therapeutic complex will exhibit substantially thesame delivery and release kinetics.

The current disclosure also provides a method of separating metal ions.The method can comprise contacting a liquid comprising a plurality ofmetal ions with a composition as described herein, under conditionssufficient to form a metal ion-composition complex comprising a metalion of the plurality of metal ions. The method can further compriseseparating a first fraction of the mixture enriched for the metalion-composition complex from a second fraction depleted for the metalion-composition complex. The first fraction can be enriched for a firstmetal ion that has a charge that is different from a charge of a secondmetal ion enriched in the second fraction.

In addition, the current disclosure provides a method of preparing Bk⁴⁺from a mixture. The method can comprise contacting a first mixturecomprising Bk⁴⁺ and a trivalent metal ion with a composition asdescribed herein under conditions sufficient to form a complexcomprising the trivalent metal ion and the composition. The method canfurther include separating the complex from the first mixture togenerate a second mixture depleted for the trivalent metal ion andchromatographically isolating the Bk⁴⁺ in the second mixture.

Further, the current disclosure provides a method of reclaiming anactinide from a sample. The method comprises obtaining an aqueous samplecomprising, or suspected of comprising, an actinide, contacting thesample with a composition as described herein to generate a mixtureunder conditions sufficient to form a complex comprising the actinideand the composition, and separating the complex from the mixture.

Many of the described benefits of the SCCs derive from use of the novelchelator and chelator combinations disclosed herein that include anumber of chelator backbone arrangements with functional groups thatcoordinate with metals. The siderocalin-metal chelator combinations canbe used to deliver radionuclides for imaging and therapeutic purposes.

Finally, disclosed herein are efficient chelator and SCC manufacturingprocesses.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Complexation of M(III) and M(IV) by the hexadentate siderophoreenterobactin or the octadentate synthetic analogs 3,4,3-LI(CAM) or3,4,3-LI(1,2-HOPO), when deprotonated.

FIG. 2. A superposition of the structures of wild-type, humansiderocalin and two mutants: T54C and S68C. Structures were determinedby x-ray crystallography previously or as described (paragraph [0248]).Siderocalin structures are shown as cartoon ribbons, highlighting theconservation of overall structure despite mutagenesis. The side-chainsof four residues are highlighted in licorice-stick representations:T/C54, S/C68, W79, and R81.

FIGS. 3A and 3B. 3,4,3-LI(2,2-diphenylbenzo[d][1,3]-2,3-catecholamide)(5)—NMR Spectra.

FIGS. 4A and 4B. 3,4,3-LI(CAM) (6)—NMR Spectra.

FIG. 5. 3,4,3-LI(CAM) (6)—Mass Spectrum, Positive Mode.

FIGS. 6A and 6B. (6A) Example of spectrophotometric competitiontitration of Th(IV)-CAM complexes. Starting conditions: 50 μM3,4,3-LI(CAM), 50 μM Th(IV), 112 μM DTPA, 3 mM CHES, 3 mM TRIS, 3 mMMES, 10 mM HCl. I=0.1 M (KCl). T=25° C. 130 spectra measured between pH2.4 and 11.5. Path length=10 mm. Spectra corrected for dilution. Inset:Change in absorbance 360 nm (squares), 340 nm (crosses), 280 nm(circles) and 265 nm (diamonds) as a function of pH. (6B) Speciationdiagram of the 3,4,3-LI(CAM) ligand in the presence of Th(IV).[Th]=[CAM]=10 μM. T=25° C., I=0.1 M. Species: CAMH₈, CAMH₇ ⁻ ,[CAMHTh]³⁻ and [CAMTh]⁴⁻. Calculations performed with Hyss software.

FIGS. 7A and 7B. (7A) Example of spectrophotometric competitiontitration of Zr(IV)-CAM complexes. Starting conditions: 50 μM3,4,3-LI(CAM), 50 μM Zr(IV), 56 μM DTPA, 5 mM CHES, 5 mM TRIS, 5 mM MES,45 mM HCl. I=0.1 M (KCl). T=25° C. 230 spectra measured between pH 1.4and 11.8. Path length=10 mm. Spectra corrected for dilution. Inset:Change in absorbance 360 nm (squares), 340 nm (crosses) and 265 nm(diamonds) as a function of pH. (7B) Speciation diagram of the3,4,3-LI(CAM) ligand in the presence of Zr(IV). [Zr]=[CAM]=10 μM. T=25°C., I=0.1 M. Species: CAMH₈, CAMH₇ ⁻ , [CAMHZr]³⁻ and [CAMZr]⁴⁻.Calculations performed with Hyss software.

FIGS. 8A and 8B. (8A) Example of spectrophotometric competitiontitration of Eu(III)-CAM complexes. Starting conditions: 50 μM3,4,3-LI(CAM), 50 μM Eu(IV), 10 mM CHES, 10 mM MES, 10 mM acetic acid,10 mM HCl. I=0.1 M (KCl). T=25° C. 215 spectra measured between pH 2.0and 11.9. Path length=10 mm. Spectra corrected for dilution. Inset:Change in absorbance 360 nm (squares), 340 nm (crosses) and 265 nm(diamonds) as a function of pH. (8B) Speciation diagram of the3,4,3-LI(CAM) ligand in the presence of Eu(III). [Eu]=[CAM]=10 μM. T=25°C., I=0.1 M. Species: CAMH₈, CAMH₇ ⁻ , CAMH₆ ²⁻, [CAMH₂Eu]³⁻, [CAMHEu]⁴⁻and [CAMEu]⁵⁻. Calculations performed with Hyss software.

FIG. 9. Crystallography data collection and refinement statistics.

FIG. 10. Synthesis of 3,4,3-LI(CAM). (A) H₂SO₄, MeOH, 65° C., 16 h(88%). (B) dichlorodiphenylmethane, 160° C., 1 h. (C) 50/50 THF/H₂O,reflux 5 h (81% over 2 steps). (D) (COCl)₂, toluene, cat. DMF; thenspermine, Et₃N, THF, 50° C., O/N (78%). (E) AcOH/H₂O+conc. HCl, 16 h(90%).

FIG. 11. Protonation and Eu(III), Zr(IV), and Th(IV) Complex FormationConstants for 3,4,3-LI(CAM)^(a a)/=0.1 M (KCl), T=25° C. Errorscorrespond to standard deviations from at least three independenttitrations. Protonation and Eu(III), Zr(IV), and Th(IV) complexformation constants previously reported for 3,4,3-LI(1,2-HOPO) are alsogiven for comparison.

FIGS. 12A and 12D. Scn dissociation constants determined fromfluorescence quenching analyses for M(III) and M(IV) complexes formedwith Ent (12A, top left), 3,4,3-LI(1,2-HOPO) (12B, top right), or3,4,3-LI(CAM) (12C, bottom left), and crystal pictures for the Scnadducts formed with the Zr(IV) (12D, bottom middle) and Th(IV) (12E,bottom right) complexes of 3,4,3-LI(CAM). The charges of the discussedmetal complexes vary from 0 to −4 at pH 7.4 and are indicated above eachbar; asterisks indicate that no binding was observed. Ent and3,4,3-LI(1,2-HOPO) data are plotted based on previously reported values(Allred, et al., PNAS, 2015, 112 (33): 10342-10347), except for the Zrcomplex K_(d) values that were determined in this work; the affinityobserved with the ferric complex of Ent is shown for reference, as it isthe native Scn ligand.

FIGS. 13A-13E. Crystallographic analyses of the binding of²³²Th-3,4,3-LI(CAM) and Zr-3,4,3-LI(CAM) by Scn. (13A) A molecularsurface representation of the calyx of Scn is colored by atom type (C:light gray, O: medium gray, N: dark grey), with the²⁴³Am-3,4,3-LI(1,2-HOPO) complex shown in a licorice-stickrepresentation, also shaded by atom type. The metal ion is shown as asmall sphere, in the center of the FIG. Surface contributed by theside-chains of W79 and R81 are shaded areas I and II, respectively. Twoprominent dark grey-tipped protuberances sticking into the calyxcorrespond to the side-chains of K125 and K134, which bracket thecrucial binding pocket in the calyx. (13B) A view into the Scn calyx ofthe ²³²Th-3,4,3-LI(CAM) complex structure, shown as in (13A). In thisstructure, the side-chain of W79 is disordered, indicated by the dashedcircle, due to sampling of multiple rotamers in the complex, allowingmuch more of the surface of R81 to be seen. Only one substituent CAMgroup and the actinide (sphere in the center of the FIG) are ordered andvisualized in the complex crystal structure, as had been seen inprevious hexadentate CAM/actinide/Scn structure [Allred et al.,Proceedings of the National Academy of Sciences 2015, 112 (33),10342-10347]. The one ordered CAM moiety sits in the key binding pocketdefined by the side-chains of K125 and K134. (13C) A view into the Scncalyx of the Zr-3,4,3-LI(CAM) complex structure, shown as in (13A) and(13B). In this structure, the side-chains of W79 and R81 haverepositioned to accommodate this ligand. Like the ²³²Th structure, onlyone substituent CAM group and the Zr atom (sphere in the center of theFIG) are ordered. (13D) A view perpendicular to that in (13A), (13B),and (13C) highlights the differential packing of the CAM or HOPOsubstituents in the key binding pocket. Other atoms in the chelator havebeen removed for clarity, and the molecular surface has been renderedpartially transparent. Metal and carbon atoms have been re-shaded toindicate the complex: ²⁴³Am-3,4,3-LI(1,2-HOPO), ²³²Th-3,4,3-LI(CAM), andZr-3,4,3-LI(CAM) (13E). The side-chains of W79 and R81 and theconnecting protein backbone have been isolated and superimposed from thethree complexes, they are shown in a licorice-stick representation,shaded as in (13D). This view highlights the different rotamers selectedin the different complexes, the only element of conformationalflexibility in the extremely rigid Scn calyx.

FIG. 14. Biodistribution of Pu(IV) when administered as a free orScn-bound 3,4,3-LI(CAM) complex. Healthy mice injected intravenouslywith ²³⁸Pu-ligand and ²³⁸Pu-ligand-protein solutions (370 Bq/mouse);mice euthanized at 4, 24, or 48 h. “ART” and “Soft” stand for “abdominalremaining tissues” and other “soft tissues,” respectively.

FIG. 15. Exemplary sequences of siderocalin orthologs.

FIG. 16. Exemplary sequences of siderocalins that can be used in thechelating platforms disclosed herein.

FIG. 17 is a flow chart of some embodiments of the processes leading tothe formation of a low-molecular weight species or high-molecular weightspecies.

FIG. 18 depicts a bar graph showing the percentage of Sn(IV)-HOPOcomplex (right bar in each pair of experiments) orsiderocalin-Eu(III)-3,4,3-LI(1,2-HOPO) passed through the cut-offfilter. Left: filter from GE HealthCare, model “VivaSpin 500-3 kDa”.Right: filter from NanoSep, model “3 kDa”. Sample centrifuged 15 min at10,000 rpm. T=22° C. Tin sample: [Sn(IV)]=25 μM, [3,4,3-LI(1,2-HOPO)]=25μM, pH=7.4. Europium sample: [Eu(III)]=300 nM, [3,4,3-LI(1,2-HOPO)]=300nM, [siderocalin]=350 nM, pH=7.4.

FIG. 19. Percentage of europium passed through the cut-off filter forsamples containing Eu(III)-3,4,3-LI(1,2-HOPO) complex without (left) andwith (right) siderocalin. Sample without siderocalin: [Eu(III)]=1 μM,[3,4,3-LI(1,2-HOPO)]=1 μM, pH=7.4. Sample without siderocalin:[Eu(III)]=0.3 μM, [3,4,3-LI(1,2-HOPO)]=0.3 μM, [siderocalin]=0.35 μM,pH=7.4. Sample centrifuged 15 min at 10,000 rpm. T=22° C. Cut-off filterfrom GE HealthCare, model “VivaSpin 500-3 kDa”.

FIG. 20 depicts a scheme of the gravity column used for metal ionsseparation.

FIG. 21 is a picture of fractions collected from Sephadex G25 PD-10column under UV irradiation (λ=312 nm). Top: initial sample containingEu³⁺ ions, [3,4,3-LI(1,2-HOPO)]⁴⁻ and siderocalin. Bottom: initialsample containing Eu³⁺ ions and [3,4,3-LI(1,2-HOPO)]⁴⁻. The bracketedsection is due to the presence of the complexessiderocalin-[Eu(III)-3,4,3-LI(1,2-HOPO)] (top) or[Eu(III)-3,4,3-LI(1,2-HOPO)]⁻ (bottom) which are luminescent under UVirradiation. Each fraction is about 0.5 mL. pH=7.4. T=25° C. Sampleseluted with TBS buffer.

FIG. 22 is a graph showing the fluorescence analysis of the fractiondepicted on FIG. 21. Triangles: sample with siderocalin; [Eu(III)]=0.3μM, [3,4,3-LI(1,2-HOPO)]=0.3 μM, [siderocalin]=0.35 μM, pH=7.4.Diamonds: sample without siderocalin; [Eu(III)]=1.0 μM,[3,4,3-LI(1,2-HOPO)]=1.0 μM, pH=7.4. Gravity column: Sephadex G-25 PD-10(GE HealthCare). Samples eluted with TBS at pH 7.4. T=22° C.Fluorescence signal measure at 611 nm after excitation of the samples at325 nm.

FIG. 23 is a graph depicting the UV-vis analysis of a tin(IV) samplepassed through a Sephadex G25 PD-10 size-exclusion column. Initialsample containing Sn⁴⁺ ions and [3,4,3-LI(1,2-HOPO)]⁴⁻. The complex[Sn(IV)-3,4,3-LI(1,2-HOPO)] was detected by UV-vis at 304 nm whichcharacteristic of this complex. Each fraction is about 0.5 mL. pH=7.4.T=25° C. Sample eluted with TBS buffer.

FIG. 24 is a graph depicting the liquid scintillation analysis of a²³⁸Pu(IV) sample passed through a Sephadex G25 PD-10 size-exclusioncolumn. Initial sample containing Pu⁴⁺ ions and [3,4,3-LI(1,2-HOPO)]⁴⁻.The presence of plutonium in each fraction was controlled by liquidscintillation counting. Each fraction is about 0.5 mL. pH=7.4. T=25° C.Sample eluted with TBS buffer.

FIG. 25 is a graph depicting the analysis of the fractions collectedafter passing a sample containing ²³⁸Pu⁴⁺ ions and ²⁴⁸Cm³⁺ ions.Crosses: detection of ²⁴⁸Cm³⁺ ions by fluorescence (Fluorescence signalmeasure at 614 nm after excitation of the samples at 325 nm). Squares:liquid scintillation analysis of ²³⁸Pu. Both fluorescence and liquidscintillation signals were normalized for comparison. Initial sample:[Cm]=0.500 μM, [Pu]=0.020 μM, [3,4,3-LI(1,2-HOPO)]=0.570 μM,[siderocalin]=0.6 μM, pH=7.4. Gravity column: Sephadex G-25 PD-10 (GEHealthCare). Samples eluted with TBS at pH 7.4. T=22° C.

FIG. 26 depicts the relative chromatographic retention of[Ce^(IV)3,4,3-LI(1,2-HOPO)], [²³²Th^(IV)3,4,3-LI(1,2-HOPO)],[²⁴²Pu^(IV)3,4,3-LI(1,2-HOPO)], and [²⁴⁹Bk^(IV)3,4,3-LI(1,2-HOPO)],relative to [Zr^(IV)3,4,3-LI(1,2-HOPO)], on an XDB-C18 column. Detectionachieved by mass spectrometry (m/z=859, 909, 1001, 1011, 1018 for Zr,Ce, Th, Pu, Bk, respectively).

DETAILED DESCRIPTION

Nuclear medicine refers to the diagnosis and/or treatment of conditionsby administering radioactive isotopes (radioisotopes or radionuclides)to a subject. For example, nuclear medicine can be used to diagnosevarious conditions through the use of imaging, such as positron emissiontopography (PET) imaging. Therapeutic nuclear medicine is often referredto as radiation therapy or radioimmunotherapy (RIT). Examples ofconditions that are treated with RIT include various cancers, thyroiddiseases, blood disorders, and restenosis following balloon angioplastyand/or stent placement.

Many conditions treated with RIT are associated with uncontrolled orunwanted cell division. When dividing cells are exposed to sufficientlyhigh doses of ionizing radiation, such as in the form of alphaparticles, beta particles, or x-ray or gamma radiation, DNA strandsbreak disrupting the normal process of cell division and inhibiting oreven stopping cellular growth.

While nuclear medicine offers many effective diagnostic and therapeuticuses, there are drawbacks associated with its use. One drawback is thatsites of the body other than the area of diagnostic or therapeuticinterest are affected by the radioactivity, often leading to unwantedside effects. This drawback is caused by release of radioactivity beforearrival at a site of interest.

Attempts have been made to lessen the release of radioactivity beforearrival at a physiological site of interest. In one approach,radionuclides have been attached to chelating agents that are thenattached to a targeting ligand that specifically recognizes and attachesto particular cell types. A common example of such a targeting ligand isan antibody. For example, antibodies targeted to tumor-specific cellsurface markers or other disease-related markers have been chemicallyconjugated to potent synthetic chelating agents such as DOTA(1,4,7,10-tetra-azacylcododecane-N,N′,N″,N′″-tetraacetic acid) and DTPA(diethylenetriamine pentaacetic acid). These chelating agents have thenbeen charged with radioisotopes of the rare earth elements such as Y³⁺or Lu³⁺ or similar trivalent metal ions, such as In³⁺ or Bi³⁺. At leasttwo such radionuclide-conjugated antibodies directed against thetumor-specific cell surface marker, CD20, have been approved for use inhuman patients: Zevalin® (RIT Oncology, LLC, Seattle, Wash.) and Bexxar®(GlaxoSmithKline, LLC, Wilmington, Del.).

U.S. Patent Publication No. 2011/0262353 (Skerra) describes drawbacks ofthe use of antibody-chelator combinations to deliver radionuclides.Skerra notes long circulation times, leading to reduced site-specificdelivery and lowered therapeutic effectiveness and contrast for imaging.Skerra suggests that a solution to this perceived problem would be tocouple targeting ligands that are smaller than antibodies to proteinsthat bind to metal chelators, such as siderocalin (Scn). Scn, also knownas Lipocalin-2 or neutrophil gelatinase-associated lipocalin, is amember of the lipocalin family of proteins that binds siderophores, atype of small chelator, with very high affinity (in the sub-nanomolarrange).

Skerra particularly teaches modifying natural Scn to form Scn “muteins”that bind non-natural Scn ligands. Skerra defines non-natural ligands asany compound which does not bind to native, mature hNGAL underphysiological conditions. See, for example, Skerra, paragraph [0021].Thus, non-natural ligands exclude many, if not all, metal and chelatedmetal complexes. More particularly, following alteration of the naturalScn protein, the Scn muteins bind non-natural ligands that Scn does notbind with under normal physiological conditions. The alterations to Scninclude mutations at one or more of positions 33, 36, 41, 52, 54, 68,70, 79, 81, 134, 136 and 138. While Skerra's approach increases Scn'sability to bind to targeting ligands that are non-natural Scn bindingpartners, this approach generates other issues with the targeteddelivery of radionuclides described more fully below. Further, even withtargeted delivery, the stability of the radioactive complex (chelatorand radionuclide) is often not strong enough to sufficiently preventearly release of metallic radioisotopes in unintended areas of the body,creating off-target side effects.

There are other challenges associated with the use of nuclear medicine.For example, one beneficial use of nuclear medicine would be toadminister a targeted radioactive imaging complex to ensure that theradioactive complex selectively reaches the physiological site ofinterest. Once selective delivery is confirmed with the imaging complex,a radioactive therapeutic complex could be administered. In practice,however, such targeted delivery of a therapeutic cannot be confirmedwith pre-imaging. This is because of two reasons. First, differentradionuclides are used for imaging and therapeutic purposes and thereare currently no “universal chelators” that can bind all types ofmetallic radionuclides. When different chelators must be used for animaging radionuclide versus a therapeutic radionuclide, the activity ofone within the physiological environment is not sufficiently predictiveof the activity of the second. Second, currently available chelators donot effectively shield radionuclides from the physiological environment.This means that radionuclides interact with the physiologicalenvironment following administration and en route to a site of interest.As a result, radionuclides with different charge states (²⁺, ³⁺, ⁴⁺)interact with the physiological environment differently, potentiallyaffecting intended delivery.

Finally, there are significant challenges associated with the efficientmanufacturing and use of ionizing radiation in therapeutic and imagingenvironments.

The present disclosure provides siderocalin (Scn)-metal chelatorcombinations that bind metallic radioisotopes used in nuclear medicinewith high affinity and effectively shield the metallic radioisotopesfrom the physiological environment. The high affinity siderocalin-metalchelator combinations include a number of backbone arrangements withfunctional groups that coordinate with metals. The siderocalin-metalchelator combinations can be used to deliver radionuclides for imagingand therapeutic purposes. These disclosed chelating platforms providenumerous benefits.

First, like Skerra, the disclosed chelating platforms utilize Scn.However, the current disclosure teaches that the muteins described inSkerra, designed to increase binding to non-natural ligands (e.g.,targeting ligands) have reduced chelating efficacy. Thus, Scn utilizedin the currently disclosed Scn-metal chelator combinations do notinclude mutations that reduce chelating efficacy. If Scn mutations areused, the mutations maintain or increase, rather than decrease, Scn'schelating efficacy.

Second, the Scn-metal chelator combinations disclosed herein have highaffinity, both between the Scn and metal chelator and between the metalchelator and radionuclide. The high affinity between each of thesecomponents reduces early release of radioactivity, reducing side effectsassociated with the use of nuclear medicine.

Third, in particular embodiments, the current disclosure provides auniversal chelating platform that accommodates metallic radioisotopesused in nuclear medicine and effectively shields them from thephysiological environment following administration. As is understood byone of ordinary skill in the art, effective shielding can be confirmedby thermodynamic and kinetic assays. By accommodating and effectivelyshielding metallic radioisotopes used in nuclear medicine, the Scn-metalchelator combinations (sometimes referred to as SCCs herein) can be usedto administer a targeted radioactive imaging complex to ensure that theradioactive complex selectively reaches the physiological site ofinterest. Once selective delivery is confirmed with the imaging complex,a radioactive therapeutic complex can be administered, with confidencethat the radioactive therapeutic complex will exhibit substantially thesame delivery and release kinetics.

Many of the described benefits of the Scn-metal chelator combinationsderive from use of the novel chelator and chelator combinationsdisclosed herein that include a number of backbone arrangements withfunctional groups that coordinate with metals. Finally, disclosed hereinare chelator and SCC manufacturing processes.

Aspects of the disclosure are now described in more detail.

Lipocalins/Siderocalin. Lipocalins are extracellular ligand-bindingproteins that are found in a variety of organisms from bacteria tohumans. Lipocalins possess many different functions, such as the bindingand transport of small hydrophobic molecules, nutrient transport, cellgrowth regulation, modulation of the immune response, inflammation, andprostaglandin synthesis. Lipocalins have a deep ligand-binding pocket,which allows for high-affinity ligand interactions. Ligands that bindlipocalin can become deeply buried within the protein, and can thereforebe shielded from the physiological environment.

Siderocalin (Scn), also known as Lipocalin-2 or neutrophilgelatinase-associated lipocalin, is a member of the lipocalin familythat binds siderophores, a type of small chelator, with very highaffinity (in the sub-nanomolar range). Siderophores secreted by microbescan steal iron from host organisms by binding tightly to iron anddelivering the iron to the microbe. Scn secreted by host organisms canprevent iron-pirating by microbes, by sequestering siderophores andpreventing their delivery back to the microbe. Therefore, high affinitybinding to chelators is a natural function of Scn.

Scn also has an exceptionally stable protein structure, and therefore isan ideal binding partner for fusion proteins, as the stability of theScn domain can impart stability on the whole fusion protein.Additionally, Scn naturally contains a secretion signal, so Scn can be auseful fusion partner for of a variety of peptides, proteins, andprotein domains, including when extracellular expression is desired.Further, Scn possesses a single N-linked glycosylation site, which isinvolved in correct processing in the ER before secretion. Anotheradvantage is that human Scn can be used, reducing stimulation of immuneresponses against it in human diagnostic and/or therapeutic uses. Makingminimal (e.g., 3 or less or 2 or less) mutations to the Scn can alsominimize the likelihood of immune response stimulation. For all of thesereasons, Scn was chosen as the chelator binding protein for thechelating platforms disclosed herein.

In particular embodiments, Scn refers to a natural Scn sequence thatretains its natural specificity for its chelator binding partners, suchas carboxymycobactin and enterochelin. Retaining natural specificitymeans that there is no statistically significant difference in bindingaffinity when assessed under comparable conditions. In particularembodiments, Scn particularly refers to the human ortholog of Scn(SWISS-PROT Data Bank Accession Number P80188), which has 178 aminoacids and a molecular weight of 20,547 Da (FIG. 15; SEQ ID NO: 1, whichis P80188 with the first 20 amino acids deleted). In particularembodiments, Scn can refer to the ortholog expressed by another species,such as the mouse ortholog (FIG. 15; SEQ ID NO: 7, which is SWISS-PROTData Bank Accession Number P11672 with the first 20 amino acidsdeleted), or the rat ortholog (FIG. 15; SEQ ID NO: 8, which isSWISS-PROT Data Bank Accession Number P30152 with the first 20 aminoacids deleted). For additional orthologs, see FIG. 15; SEQ ID NOs. 2-6and 9-18) and Correnti & Strong, (2013) “Iron Sequestration in Immunity”In Metals in Cells, Encyclopedia of Inorganic and BioinorganicChemistry. (Culcotta & Scott, eds.) John Wiley & Sons, pp. 349-59.

In particular embodiments, Scn refers to an Scn sequence that caninclude mutations, so long as the mutations do not significantly affectScn's natural specificity for its chelator binding partners, such ascarboxymycobactin and enterochelin. Human Scn residues that areimportant for binding to siderophores include (referring to SEQ ID NO:1): 52, 54, 68, 70, 79, 81, 100, 106, 123, 125, 132, 133, 134, 138, and141. Holmes, et al., Structure 2005, 13, 29-41. In particularembodiments, Scns disclosed herein exclude mutations at one or more ofresidues: 52, 54, 68, 70, 79, 81, 100, 106, 123, 125, 132, 133, 134,138, and 141 of SEQ ID NO: 1. In particular embodiments, Scns disclosedherein exclude mutations at 2 or more, 3 or more, 4 or more, 5 or more,6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12or more, 13 or more, 14 or more or 15 or more of residues: 52, 54, 68,70, 79, 81, 100, 106, 123, 125, 132, 133, 134, 138, and 141 of SEQ IDNO: 1. In particular embodiments, Scns disclosed herein excludemutations at residues: 52, 54, 68, 70, 79, 81, 100, 106, 123, 125, 132,133, 134, 138, and 141 of SEQ ID NO: 1.

In particular embodiments, Scns can include mutations that improve itsability to bind to chelators. For example, mutations at residues 54and/or 68 can stabilize Scn interaction with chelators by providing a“chelator trap”. In particular embodiments, chelator traps can becreated by providing a cysteine at residues 54 and/or 68 to introduce athiol moiety. FIG. 2. shows the superposition of the structures ofwild-type, human Scn and two mutants: T54C and S68C. Scn structures areshown as cartoon ribbons, highlighting the conservation of overallstructure despite mutagenesis. The side-chains of four residues arehighlighted in licorice-stick representations: T/C54, S/C68, W79, andR81. W79 and R81 are the most mobile side-chains in the ligand bindingsite, and are observed in different rotamers in different structures.The side-chains at positions 54 and 68 show that replacing the wild-typeresidues with cysteine does not affect the local structure of the ligandbinding site, conserving wild-type ligand specificity. The crystalstructures of the mutants, protected deep in the ligand binding site,also show that these cysteines, protected deep in the ligand bindingsite, remain reduced, available for conjugation.

In particular embodiments, at least one of the amino acids of Scn may besubstituted for a non-native amino acid to achieve a different benefit.For example, a C87S mutation in Scn can prevent dimerization and canyield pure monomeric fusion protein. Goetz, et al., Molecular Cell 2002,10, 1033-43. In additional embodiments, mutations can be generated thatenable interactions with non-native ligands, so long as the mutations donot reduce chelator binding affinity. FIG. 16 depicts Scn of SEQ ID NO:1 altered to include: a T54C mutation (SEQ ID NO: 19); a S68C mutation(SEQ ID NO: 20); T54C and S68C mutations (SEQ ID NO: 21); a C87Smutation (SEQ ID NO: 22); a leader sequence, K125A and C87S mutations(SEQ ID NO: 23); a leader sequence, T54C, S68C, C87S, and K125Amutations (SEQ ID NO: 24); and a leader sequence, T54C and C87Smutations, a GSS linker, and a CD19 scFV (SEQ ID NO: 25). In particularembodiments, any combination of mutations depicted in FIG. 16 can beused. Additional potential alternations to Scn that can be used aredescribed in the Section of this application related to Methods ofProtein Expression.

Chelators. Chelators are molecules that can bind metals. Chelators caninclude organic molecules that covalently bond with a metal. As usedherein, a covalent bond describes the sharing of one or more pairs ofelectrons between atoms. In some instances, chelators are agents thatremove excess metal from an environment. Previous metal chelators thathave been joined to proteins for the purpose of loading the chelatorwith a radioisotope are described in U.S. Pat. Nos. 4,454,106;4,472,509; 4,831,175; 5,246,692; 5,250,285; 5,514,363; 5,837,218;5,891,418; 5,922,302; 6,180,082; 6,183,721; and 6,203,775.

In particular embodiments, chelators can include a number of metalcoordinating atoms that bond with a metal. The metal coordinating atomscan bond with metals having cations with a +1 charge. The metalcoordinating atoms can also bond with metals having cations with a +2charge. Additionally, the metal coordinating atoms can bond with metalshaving cations with a +3 charge. Further, the metal coordination atomscan bond with metals having cations with a +4 charge. The chelatorsdescribed herein can, in some cases, include siderophores.

In particular embodiments, the metal coordinating atoms of the chelatorsdescribed herein can be included in one or more functional groups of thechelators. In some examples, the metal coordinating atoms of thechelators can be included in one or more catecholate (CAM) groups. A CAMgroup can include at least a phenyl ring substituted by hydroxyl groupson adjacent carbon atoms. According to some illustrative embodiments, aCAM group can include:

In particular embodiments, the metal coordinating atoms of the chelatorscan be included in one or more hydroxymate (HA) groups. According tosome embodiments, a HA group can include:

where R_(a) can include H or an alkyl group including no greater than 5carbon atoms. For example, R_(a) can include a methyl group, an ethylgroup, a propyl group, an isopropyl group, a butyl group, a sec-butylgroup, an iso-butyl group, a tert-butyl group, a pentyl group, atert-pentyl group, a neopentyl group, or an iso-pentyl group.

In particular embodiments, the metal coordinating atoms of the chelatorscan be included in one or more hydroxypyridinone (HOPO) groups. A HOPOgroup can include a pyridinone ring substituted by a hydroxyl group onthe N atom. In some cases, a HOPO group can include a 1,2-HOPO group.According to some illustrative embodiments, a 1,2-HOPO group caninclude:

The metal coordinating atoms of the chelators can be included incombinations of two or more of one or more CAM groups, one or more HAgroups, or one or more HOPO groups. In illustrative examples, the metalcoordinating atoms of the chelators can be included in one or more CAMgroups and one or more HA groups. In other illustrative examples, themetal coordinating atoms of the chelators can be included in one or moreCAM groups and one or more HOPO groups. In additional illustrativeexamples, the metal coordinating atoms of the chelators can be includedin one or more HA groups and one or more HOPO groups. In furtherillustrative examples, the metal coordinating atoms of the chelators canbe included in one or more HA groups, one or more CAM groups, and one ormore HOPO groups.

The chelators can include a number of functional groups having metalcoordinating atoms with the functional groups being bonded to a linearscaffold or a branched scaffold. The functional groups and/orsubstituents described herein may be substituted or unsubstituted.Substituted functional groups and/or substituents can be substituted byone or more hydroxyl groups, one or more alkyl groups having no greaterthan 10 carbon atoms, one or more amine groups, one of more thiolgroups, one or more ester groups, or combinations thereof.

The scaffold can include one or more amine groups. An amine group caninclude a nitrogen atom bonded to three substituents. In particularembodiments, an amine group can include a nitrogen atom bonded at leastone carbon atom of substituent. In various embodiments, an amine groupcan include a nitrogen atom bonded to at least a first carbon atom of afirst substituent and a second carbon atom of a second substituent. Infurther embodiments, an amine group can include a nitrogen atom bondedto a first carbon atom of a first substituent, a second carbon atom of asecond substituent and a third carbon atom of a third substituent. Incertain embodiments, an amine group can include a nitrogen atom bondedto one or more hydrogen atoms.

In some embodiments, the scaffold can include one or more amide groups.An amide group can include a nitrogen atom bonded to a carbonyl groupand two additional substituents. In various examples, an amide group caninclude a nitrogen atom bonded to a carbonyl group and a carbon atom ofa first additional substituent. In other examples, an amide group caninclude a nitrogen atom bonded to a carbonyl group and a first carbonatom of a first additional substituent and a second carbon atom of asecond additional substituent.

In particular embodiments, the scaffold can include one or more aminegroups and one or more amide groups. The scaffold can include one ormore carbon-based chains bonded between amine groups, a carbon-basedchain bonded between amide groups, or one or more carbon-based chainsbonded between a combination of one or more amine groups and one or moreamide groups. The carbon-based chains can include at least one carbonatom, at least 2 carbon atoms, at least 3 carbon atoms, at least 4carbon atoms, or at least 5 carbon atoms. In addition, the carbon-basedchains can include no greater than 10 carbon atoms, no greater than 9carbon atoms, no greater than 8 carbon atoms, no greater than 7 carbonatoms, or no greater than 6 carbon atoms. In various embodiments, thecarbon-based chains can include from 1 carbon atom to 10 carbon atoms,from 2 carbon atoms to 7 carbon atoms, or from 3 carbon atoms to 6carbon atoms. In illustrative embodiments, the carbon-based chains caninclude alkane chains having carbon-carbon single bonds. In some cases,the carbon-based chains can include alkene chains having at least onecarbon-carbon double bond. The carbon-based chains can be substituted orunsubstituted.

In particular embodiments, compositions that function as chelators forradionuclides can have the following structure, referred to herein asStructure I:

In some examples, A1, A2, A3, and A4 can, individually, include a CAMgroup, a HOPO group, or a HA group. Additionally, B1, B2, B3, and B4can, individually, include an amide group or an amine group. Further, atleast one of C1, C2, C3, C4, C5, or C6 can, individually, include SH,C(═O)OH, or NH₂. Also, in various examples, at least another one of C1,C2, C3, C4, C5, or C6 can be optional. In particular examples, at leastone of L1, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11, L12, or L13 can,individually, include H, an alkyl group having no greater than 10 carbonatoms, an alkylamino group having no greater than 10 carbon atoms and nogreater than 2 nitrogen atoms; an alkyl ether group having no greaterthan 10 carbon atoms, a hydroxy ester group, or an alkyl ester grouphaving no greater than 10 carbon atoms. In certain examples, at leastone of L1, L5, L6, L7, L8, L9, L10, L11, L12, or L13 can be optional.

In illustrative examples, at least another one of L2, L3, or L4, can,individually, include an amine group or an amide group. In additionalillustrative examples, L1, C1, L7, C2, L9, C3, L11, C4, and L13, C5 canbe absent, L5 can include an alkyl group having no greater than 5 carbonatoms, and C6 can include SH, C(═O)OH, or NH₂. In further illustrativeexamples, L2, L3, L4, L6, L8, L10, and L12 can, individually, include analkyl group having no greater than 5 carbon atoms. Also, A1 can includea CAM group or a 1, 2-HOPO group; A2 can include a HA group, A3 caninclude a HA group, and A4 can include a CAM group, a 1,2-HOPO group, ora HA group. In other illustrative examples, at least one of L2, L3, orL4 includes an alkylamino group.

In various illustrative examples, B1, B2, and B3 can, individually,include an amide group and B4 can include an amino group, L2 and L3 caninclude an amino group, and L4 can include an alky group having nogreater than 5 carbon atoms. Additionally, C1, C2, C3, C4, C5, L1, A1,A2, A3, L1, L6, L7, L8, L9, L10, L11, L12, and L13 can be absent; A4 caninclude a CAM group, a 1,2-HOPO group, or a HA group; and L5 can includean alkyl group having no greater than 5 carbon atoms.

In certain illustrative examples, 1, B2, and B3 can include an amidegroup and B4 can include an amide group; L2 and L3 can, individually,include an amino group; and L4 includes an alky group having no greaterthan 5 carbon atoms. Further, C1, C2, C3, C4, C5, A1, A2, A3, L1, L6,L7, L8, L9, L10, 11, and L13 can be absent, L12 can include an aminogroup, L5 can include an ether group having no greater than 10 carbonatoms, and A4 can include a CAM group, a 1,2-HOPO group, or a HA group.

In particular illustrative examples, C1, C2, C5, C6, L1, L2, L3, L4, L5,L7, L13, B2, and B4 can be absent; 1 and B3 can, individually, includean amide group; L6, L8, L10, and L12 can, individually, include an aminogroup, A1, A2, A3, and A4 can, individually, include a CAM group, a1,2-HOPO group, or a HA group; and L9 and L11 can, individually, includean alkyl group having no greater than 5 carbon atoms.

In particular embodiments, compositions that function as chelators forradionuclides can have the following structure, referred to herein asStructure II:

R₁, R₂, R₃, R₄, and R₅ can, individually, include H, an alkyl grouphaving from 1 to 10 carbon atoms, a CAM group, a HA group, or a 1,2-HOPOgroup. R₆ can include H, an alkyl group having from 1 to 10 carbonatoms, or an alkyl group having from 1 to 10 carbon atoms andsubstituted by at least one of SH, NH₂, or C(═O)OH. m can be from 1 to6; n can be from 1 to 6; and o can be from 1 to 6. In particularembodiments, at least one of R₁, R₂, R₃, R₄, or R₅ can, individually,include a CAM group, a HA group, or a 1,2-HOPO group. In variousembodiments, Structure II can include a linear, spermine-based backbone.

In particular embodiments, compositions that function as chelators forradionuclides can have the following structure, referred to herein asStructure III:

At least one of R₁, R₃, R₄, or R₅ can, individually, include a CAMgroup, a HA group, or a 1,2-HOPO group. Optionally, another one of R₁,R₃, R₄, or R₅ can, individually, include H, OH, or an alkyl group havingfrom 1 to 10 carbon atoms. R₂ can include H, OH, or an alkyl groupincluding from 1 to 5 carbon atoms. p can be from 0 to 4. R₇ can includeSH, C(═O)OH, or NH₂. In illustrative embodiments, R₁ can include a CAMgroup or a 1,2-HOPO group, R₃ and R₄ can, individually, include a HAgroup, and R₅ can include a CAM group, a 1,2-HOPO group, or a HA group.

In particular embodiments, compositions that function as chelators forradionuclides can have the following structure, referred to herein asStructure IV:

R₇ can include SH, NH₂, or C(═O)OH. R₂, R₈, and R₉ can, individually,include H, OH, or an alkyl group including from 1 to 5 carbon atoms. pcan be from 0 to 4.

In particular embodiments, compositions that function as chelators forradionuclides can have the following structure, referred to herein asStructure V:

R₇ can include SH, NH₂, or C(═O)OH. R₂, R₈, and R₉ can, individually,include H, OH, or an alkyl group including from 1 to 5 carbon atoms. pcan be from 0 to 4.

In particular embodiments, compositions that function as chelators forradionuclides can have the following structure, referred to herein asStructure VI:

R₇ can include SH, NH₂, or C(═O)OH. R₂, R₈, R₉, and R₁₀ can,individually, include H, OH, or an alkyl group including from 1 to 5carbon atoms. p can be from 0 to 4.

In particular embodiments, compositions that function as chelators forradionuclides can have the following structure, referred to herein asStructure VII:

R₇ can include SH, NH₂, or C(═O)OH. R₂, R₈, and R₉ can, individually,include H, OH, or an alkyl group including from 1 to 5 carbon atoms. pcan be from 0 to 4.

In particular embodiments, compositions that function as chelators forradionuclides can have the following structure, referred to herein asStructure VIII:

R₇ can include SH, NH₂, or C(═O)OH. R₂, R₈, and R₉ can, individually,include H, OH, or an alkyl group including from 1 to 5 carbon atoms. pcan be from 0 to 4.

In particular embodiments, compositions that function as chelators forradionuclides can have the following structure, referred to herein asStructure IX:

R₇ can include SH, NH₂, or C(═O)OH. R₂, R₈, R₉, and R₁₀ can,individually, include H, OH, or an alkyl group including from 1 to 5carbon atoms. p can be from 0 to 4.

In particular embodiments, compositions that function as chelators forradionuclides can have the following structure, referred to herein asStructure X:

In particular embodiments, compositions that function as chelators forradionuclides can have the following structure, referred to herein asStructure XI:

In particular embodiments, compositions can have a branched backbonerather than the linear, spermine-based backbone of Structures III-XI. Inparticular embodiments, compositions that function as chelators forradionuclides can have the following structure, referred to herein asStructure XII:

At least one of R₁₁, R₁₂, R₁₃, or R₁₅, can, individually, include a CAMgroup, a HA group, or a 1,2-HOPO group. Optionally, at least another oneof R₁₁, R₁₂, R₁₃, or R₁₅ can, individually, include H, OH, or an alkylgroup having from 1 to 10 carbon atoms. R₁₇ can include SH, NH₂, orC(═O)OH. r can be from 0 to 6. R₂, R₁₄, and R₁₆ can, individually,include H, OH, or an alkyl group having from 1 to 10 carbon atoms. Inillustrative embodiments, R₁₁ can include a CAM group or a 1,2-HOPOgroup, R₁₂ and R₁₅ can, individually, include a HA group, and R₁₃ caninclude a CAM group, a 1,2-HOPO group, or a HA group.

In particular embodiments, compositions that function as chelators forradionuclides can have the following structure, referred to herein asStructure XIII:

R₂, R₁₄, R₁₆, R₁₈, and R₁₉ can, individually, include H, OH, or an alkylgroup having from 1 to 10 carbon atoms. R₁₇ can include SH, NH₂, orC(═O)OH. r can be from 0 to 4.

In particular embodiments, compositions that function as chelators forradionuclides can have the following structure, referred to herein asStructure XIV:

R₂, R₁₄, R₁₆, R₁₈, and R₁₉ can, individually, include H, OH, or an alkylgroup having from 1 to 10 carbon atoms. R₁₇ can include SH, NH₂, orC(═O)OH. r can be from 0 to 4.

In particular embodiments, compositions that function as chelators forradionuclides can have the following structure, referred to herein asStructure XV:

R₂, R₁₄, R₁₆, R₁₈, R₁₉, and R₂₀ can, individually, include H, OH, or analkyl group having from 1 to 10 carbon atoms. R₁₇ can include SH, NH₂,or C(═O)OH. r can be from 0 to 4.

In particular embodiments, compositions that function as chelators forradionuclides can have the following structure, referred to herein asStructure XVI:

R₂, R₁₄, R₁₆, R₁₈, R₁₉, and R₂₀ can, individually, include H, OH, or analkyl group having from 1 to 10 carbon atoms. R₁₇ can include SH, NH₂,or C(═O)OH. r can be from 0 to 4.

In particular embodiments, compositions that function as chelators forradionuclides can have the following structure, referred to herein asStructure XVII:

R₂, R₁₄, R₁₆, R₁₈, and R₁₉ can, individually, include H, OH, or an alkylgroup having from 1 to 10 carbon atoms. R₁₇ can include SH, NH₂, orC(═O)OH. r can be from 0 to 4.

In particular embodiments, compositions that function as chelators forradionuclides can have the following structure, referred to herein asStructure XVIII:

R₂, R₁₄, R₁₆, R₁₃, and R₁₉ can include H, OH, or an alkyl group havingfrom 1 to 10 carbon atoms. R₁₇ can, individually, include SH, NH₂, orC(═O)OH. r can be from 0 to 4.

In particular embodiments, compositions can have a backbone thatincludes a number of amide groups and a number of amine groups. In someembodiments, the backbone of compositions that function as chelators forradionuclides can be based on Desferrioxamine B. In particularembodiments, compositions that function as chelators can have thefollowing structure, referred to herein as Structure XIX:

R₂₁ and R₂₂ can include H, OH, or an alkyl group having from 1 to 10carbon atoms. R₂₃ can include H, OH, an alkyl group having from 1 to 10carbon atoms, or (CH₂)_(e)R_(a), where R_(a) is SH, C(═O)OH, or NH₂ ande is from 1 to 10. R₂₄ can include a substituent that includes a CAMgroup, a 1,2-HOPO group, or a HA group. Optionally, R₂₄ can include SH,C(═O)OH, or NH₂. a, b, and c can include from 1 to 10 and d can includefrom 1 to 4.

In particular embodiments, compositions that function as chelators forradionuclides can have the following structure, referred to herein asStructure XX:

R₂₅, R₂₆, and R₂₇ can, individually, include H, OH, or an alkyl grouphaving from 1 to 10 carbon atoms. R₂₈ can include H, an alkyl grouphaving from 1 to 5 carbon atoms, SH, NH₂, or C(═O)OH. s can be from 0 to4.

In particular embodiments, compositions that function as chelators forradionuclides can have the following structure, referred to herein asStructure XXI:

R₂₅, R₂₆, R₂₇, and R₃₀ can, individually, include H, OH, or an alkylgroup having from 1 to 10 carbon atoms. R₂₈ and R₂₉ can, individually,include H, an alkyl group having from 1 to 5 carbon atoms, SH, NH₂, orC(═O)OH. s can be from 0 to 4. t can be from 0 to 4.

In particular embodiments, compositions that function as chelators forradionuclides can have the following structure, referred to herein asStructure XXII:

R₂₅, R₂₆, R₂₇, and R₃₀ can, individually, include H, OH, or an alkylgroup having from 1 to 10 carbon atoms. R₂₈ and R₂₉ can, individually,include H, an alkyl group having from 1 to 5 carbon atoms, SH, NH₂, orC(═O)OH. s can be from 0 to 4. t can be from 0 to 4.

In particular embodiments, compositions that function as chelators forradionuclides can have the following structure, referred to herein asStructure XXIII:

R₂₅, R₂₆, R₂₇, R₃₀, and R₃₁ can, individually, include H, OH, or analkyl group having from 1 to 10 carbon atoms. R₂₈ and R₂₉ can,individually, include H, an alkyl group having from 1 to 5 carbon atoms,SH, NH₂, or C(═O)OH. s can be from 0 to 4. t can be from 0 to 4.

In particular embodiments, compositions that function as chelators forradionuclides can have the following structure, referred to herein asStructure XXIV:

R₂₅, R₂₆, and R₂₇ can, individually, include H, OH, or an alkyl grouphaving from 1 to 10 carbon atoms. R₂₈ can include H, an alkyl grouphaving from 1 to 5 carbon atoms, SH, NH₂, or C(═O)OH. s can be from 0 to4.

In particular embodiments, compositions that function as chelators forradionuclides can have the following structure, referred to herein asStructure XXV:

R₂₅, R₂₆, R₂₇, and R₃₂ can, individually, include H, OH, or an alkylgroup having from 1 to 10 carbon atoms. R₂₈ can include H, an alkylgroup having from 1 to 5 carbon atoms, SH, NH₂, or C(═O)OH. s can befrom 0 to 4.

In particular embodiments, compositions can have an amide-basedbackbone. In particular embodiments, compositions that function aschelators can have the following structure, referred to herein asStructure XXVI:

A, B, C, and D can, individually, include one or more amide groups, oneor more amine groups, or an alkyl group having from 1 to 10 carbonatoms. R₃₃, R₃₄, R₃₅, and R₃₆ can, individually, include a CAM group, a1,2-HOPO group, or a HA group. g, h, i, and j can, individually, be from1 to 10.

In particular embodiments, compositions that function as chelators forradionuclides can have the following structure, referred to herein asStructure XXVII:

R₃₇ and R₄₂ can, individually, include H, an alkyl group having from 1to 5 carbon atoms, SH, C(═O)OH, or NH₂. R₃₈, R₃₉, R₄₀, and R₄₁ can,individually, include H, OH, or an alkyl group having from 1 to 5 carbonatoms. u and v can, individually, be from 0 to 5.

In particular embodiments, compositions that function as chelators forradionuclides can have the following structure, referred to herein asStructure XXVIII:

R₃₇ and R₄₂ can, individually, include H, an alkyl group having from 1to 5 carbon atoms, SH, C(═O)OH, or NH₂. R₃₈, R₃₉, R₄₀, and R₄₁ can,individually, include H, OH, or an alkyl group having from 1 to 5 carbonatoms. u and v can, individually, be from 0 to 5.

In particular embodiments, compositions that function as chelators forradionuclides can have the following structure, referred to herein asStructure XXIX:

R₃₇ and R₄₂ can, individually, include H, an alkyl group having from 1to 5 carbon atoms, SH, C(═O)OH, or NH₂. R₃₈, R₃₉, R₄₀, and R₄₁ can,individually, include H, OH, or an alkyl group having from 1 to 5 carbonatoms. u and v can, individually, be from 0 to 5.

The chelators can bind to a protein. In particular embodiments, thechelators can bind to siderocalin. In some embodiments, the chelatorscan bind to a dye. For example, the chelators can bind to a fluorophore.Additionally, the chelators can bind to both a protein and a dye. Invarious embodiments, the chelators can bind to a metal to form achelator-metal complex that can also bind to siderocalin. The metal caninclude a radionuclide.

The chelators and/or the chelator-metal complex can have an equilibriumdissociation constant with siderocalin of no greater than 100 nanomolar(nM), no greater than 90 nM, no greater than 80 nM, no greater than 70nM, no greater than 60 nM, no greater than 50 nM, no greater than 45 nM,no greater than 40 nM, no greater than 35 nM, no greater than 30 nM, nogreater than 25 nM, no greater than 20 nM, or no greater than 15 nM. Inaddition, the chelators can have an equilibrium dissociation constantwith siderocalin of at least 0.1 nM, at least 0.5 nM, at least 0.8 nM,at least 1 nM, at least 1.5 nM, at least 2 nM, at least 3 nM, at least 5nM, at least 8 nM, at least 10 nM, or at least 12 nM. It will beappreciated that the equilibrium dissociation constant between achelator and siderocalin can be within a range between any of the valuesnoted above. In illustrative examples, the equilibrium dissociationconstant between a chelator and siderocalin can be from 0.1 nM to 50 nM.In other illustrative examples, the equilibrium dissociation constantbetween a chelator and siderocalin can be from 1 nM to 40 nM. Inadditional illustrative examples, the equilibrium dissociation constantbetween a chelator and siderocalin can be from 0.8 nM to 10 nM. Infurther illustrative examples, the equilibrium dissociation constantbetween a chelator and siderocalin can be from 0.5 nM to 5 nM.

In particular embodiments, a composition E can be bound to a dye, aprotein, or both a dye and a protein. Additionally, the composition Ecan be bound to one or more CAM groups, one or more 1,2-HOPO groups, oneor more HA groups, or a combination thereof. In some embodiments, thecomposition E can be bound to the one or more CAM groups, the one ormore 1,2-HOPO groups, or the one or more HA groups through an amidelinkage. In particular embodiments, the composition E can include anumber of carbon atoms, a number of nitrogen atoms, and a number ofoxygen atoms. In illustrative embodiments, the composition E can includefrom 4 to 40 carbon atoms, from 1 to 20 nitrogen atoms, and from 1 to 15oxygen atoms. In various embodiments, the composition E can include from1 to 20 carbon atoms, from 1 to 8 nitrogen atoms, and from 1 to 5 oxygenatoms. In some illustrative examples, a composition according toembodiments herein can include the following structure, referred toherein as Structure XXXI:

R₄₃ and R₄₄ can, individually, include H or an alkyl group from 1 to 5carbon atoms. q can be from 0 to 4, w can be from 0 to 4, and x can befrom 0 to 4. In some embodiments, at least one of q, w, or x is 1.Additionally, y and z can, individually, be from 0 to 4. In variousembodiments, the dye and/or the protein can be bound to E via an SHgroup, an amide group, or a carboxyl group. In particular embodiments,one or more groups included in the structures described herein can bindto a protein and/or a dye. In illustrative examples, R₆, R₇, R₁₇, R₂₃,R₂₈, R₂₉, R₃₇, R₄₂, or combinations thereof, can, individually, bind toa protein and/or a dye.

In particular embodiments, the composition E can be bound to one or moreamino acids of the protein. For example, the composition E can be boundto one or more lysine residues of siderocalin, such as K125 and/or K134.In other examples, the composition E can be bound to one or more aminoacids of the protein that have been modified. To illustrate, siderocalincan be modified such that T54 and S68 are modified to cysteine residues.In these situations, the composition E can be bound to at least one ofthe modified Scn^(T54C) or Scn^(S68C) mutations.

Radionuclides. The Scn-chelator combinations (SCCs) disclosed herein arecharged with radionuclides for use in nuclear medicine. Radionuclidesthat are chemically compatible with the SCCs should be chosen.Chemically compatible means that the radionuclide, in its elemental formor ionic form should not be so reactive that it changes the structure orfunction of any component of SCCs in a way that impairs achievement ofan intended purpose.

Criteria for choosing radionuclides for a particular use in nuclearmedicine can include the type and energy of radioactive decay productyielded by the radioisotope; the half-life of decay; chemical propertiesof the atom or ion; and the biological and/or toxicological propertiesof the atom or ion.

The decay product(s) yielded by the decay of a radionuclide (alsoreferred to as a radioisotope) should be capable of interacting withcells in such a way as to inhibit or interfere with biological processesnecessary for cellular replication, or that cause the cell to undergoapoptosis. In particular embodiments, the decay product(s) should be ofsufficiently high energy, and sufficiently low mass such that they,whether particles or photons, reach and penetrate the nuclei of unwantedcells. Usually, the energy is not so high that the decay product(s)reach tissues far away from unwanted cells, or reach persons in closeproximity to a patient. However, there may be circumstances wherein highenergy and highly penetrative decay products are desirable. Inparticular embodiments, particularly useful radionuclides are those thatdecay with the emission of alpha particles, beta particles, gamma rays,positrons, x-rays, or Auger electrons. In particular embodiments,particularly useful radionuclides decay with the emission of alphaparticles.

Radionuclides with different half-lives can be chosen based on thelength of time desirable for irradiation of unwanted cells. Usually, ahalf-life of decay is not chosen if it is too short and thus, notsufficiently effective to arrest the growth of unwanted cells. Likewise,a half-life of decay that is too long is not chosen, thus avoiding thepersistence of radiation at a high level after such time that unwantedcells are substantially or completely growth inhibited. In this manner,deleterious side effects of radiation are minimized. In particularembodiments, isotopes that decay with half-lives of 3 hours to 300 daysare selected. Such isotopes can decay to negligible levels in 1 day to 4years.

Examples of radioisotopes useful in nuclear medicine include ²²⁵Ac,²²⁶Ac, ²²⁸Ac, ¹⁰⁵Ag, ¹⁰⁶mAg, ¹¹⁰mAg, ¹¹¹Ag, ¹¹²Ag, ¹¹³Ag, ²³⁹Am, ²⁴⁰Am,²⁴²Am, ²⁴⁴Am, ³⁷Ar, ⁷¹As, ⁷²As, ⁷³As, ⁷⁴As, ⁷⁶As, ⁷⁷As, ²⁰⁹At, ²¹⁰At,¹⁹¹Au, ¹⁹²Au, ¹⁹³Au, ¹⁹⁴Au, ¹⁹⁵Au, ¹⁹⁶Au, ¹⁹⁶m²Au, ¹⁹⁸Au, ¹⁹⁸mAu, ¹⁹⁹Au,²⁰⁰mAu, ¹²⁸Ba, ¹³¹Ba, ¹³³mBa, ¹³⁵mBa, ¹⁴⁰Ba, ⁷Be, ²⁰³Bi, ²⁰⁴Bi, ²⁰⁵Bi,²⁰⁶Bi, ²¹⁰Bi, ²¹²Bi, ²⁴³Bk, ²⁴⁴Bk, ²⁴⁵Bk, ²⁴⁶Bk, ²⁴⁸mBk, ²⁵⁰Bk, ⁷⁶Br,⁷⁷Br, ⁸⁰mBr, ⁸²Br, ¹¹C, ¹⁴C, ⁴⁵Ca, ⁴⁷Ca, ¹⁰⁷Cd, ¹¹⁵Cd, ¹¹⁵mCd, ¹¹⁷mCd,¹³²Ce, ¹³³mCe, ¹³⁴Ce, ¹³⁵Ce, ¹³⁷Ce, ¹³⁷mCe, ¹³⁹Ce, ¹⁴¹Ce, ¹⁴³Ce, ¹⁴⁴Ce,²⁴⁶Cf, ²⁴⁷Cf, ²⁵³Cf, ²⁵⁴Cf, ²⁴⁰Cm, ²⁴¹Cm, ²⁴²Cm, ²⁵²Cm, ⁵⁵Co, ⁵⁶Co,⁵⁷Co, ⁵⁸Co, ⁵⁸mCO, ⁶⁰Co, ⁴⁸Cr, ⁵¹Cr, ¹²⁷Cs, ¹²⁹Cs, ¹³¹Cs, ¹³²Cs, ¹³⁶Cs,¹³⁷Cs, ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ¹⁵³Dy, ¹⁵⁵Dy, ¹⁵⁷Dy, ¹⁵⁹Dy, ¹⁶⁵Dy, ¹⁶⁶Dy,¹⁶⁰Er, ¹⁶¹Er, ¹⁶⁵Er, ¹⁶⁹Er, ¹⁷¹Er, ¹⁷²Er, ²⁵⁰Es, ²⁵¹Es, ²⁵³Es, ²⁵⁴Es,²⁵⁴mEs, ²⁵⁵Es, ²⁵⁶mEs, ¹⁴⁵Eu, ¹⁴⁶Eu, ¹⁴⁷Eu, ¹⁴⁸Eu, ¹⁴⁹Eu, ¹⁵⁰mEu,¹⁵²mEu, ¹⁵⁶Eu, ¹⁵⁷Eu, ⁵²Fe, ⁵⁹Fe, ²⁵¹Fm, ²⁵²Fm, ²⁵³Fm, ²⁵⁴Fm, ²⁵⁵Fm,²⁵⁷Fm, ⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga, ⁷²Ga, ⁷³Ga, ¹⁴⁶Gd, ¹⁴⁷Gd, ¹⁴⁹Gd, ¹⁵¹Gd, ¹⁵³Gd,¹⁵⁹Gd, ⁶⁸Ge, ⁶⁹Ge, ⁷¹Ge, ⁷⁷Ge, ¹⁷⁰Hf, ¹⁷¹Hf, ¹⁷³Hf ¹⁷⁵Hf, ¹⁷⁹m²Hf,¹⁸⁰mHf, 181Hf, ¹⁸⁴Hf, ¹⁹²Hg, ¹⁹³Hg, ¹⁹³mHg, ¹⁹⁵Hg, ¹⁹⁵mHg, ¹⁹⁷Hg,¹⁹⁷mHg, ²⁰³Hg, ¹⁶⁰ mHo, ¹⁶⁶Ho, ¹⁶⁷Ho, ¹²³I, ¹²⁴I, ¹²⁶I, ¹³⁰I, ¹³²I,¹³³I, ¹³⁵I, ¹⁰⁹In, ¹¹⁰In, ¹¹¹In, ¹¹⁴mIn, ¹¹⁵mIn, ¹⁸⁴Ir, ¹⁸⁵Ir, ¹⁸⁶Ir,¹⁸⁷Ir, ¹⁸⁸Ir, ¹⁸⁹Ir, ¹⁹⁰Ir, ¹⁹⁰m²Ir, ¹⁹²Ir, ¹⁹³mIr, ¹⁹⁴Ir, ¹⁹⁴m²Ir,¹⁹⁵mIr, ⁴²K, ⁴³K, ⁷⁶Kr, ⁷⁹Kr, ⁸¹mKr, ⁸⁵mKr, ¹³²La, ¹³³La, ¹³⁵La, ¹⁴⁰La,¹⁴¹La, ²⁶²Lr, ¹⁶⁹Lu, ¹⁷⁰Lu, ¹⁷¹Lu, ¹⁷²Lu, ¹⁷⁴mLu, ¹⁷⁶mLu, ¹⁷⁷Lu, ¹⁷⁷mLu,¹⁷⁹Lu, ²⁵⁷Md, ²⁵⁸Md, ²⁶⁰Md, ²⁸Mg, ⁵²Mn, ⁹⁰Mo, ⁹³mMo, ⁹⁹Mo, ¹³N, ²⁴Na,⁹⁰Nb, ⁹¹mNb, ⁹²mNb, ⁹⁵Nb, ⁹⁵mNb, ⁹⁶Nb, ¹³⁸Nd, ¹³⁹mNd, ¹⁴⁰Nd, ¹⁴⁷Nd,⁵⁶Ni, ⁵⁷Ni, ⁶⁶Ni, ²³⁴Np, ²³⁶mNp, ²³⁸Np, ²³⁹Np, ¹⁵O, ¹⁸²Os, ¹⁸³Os,¹⁸³mOs, ¹⁸⁵Os, ¹⁸⁹mOs, ¹⁹¹Os, ¹⁹¹mOs, ¹⁹³Os, ³²P, ³³P, ²²⁸Pa, ²²⁹Pa,²³⁰Pa, ²³²Pa, ²³³Pa, ²³⁴Pa, ²⁰⁰Pb, ²⁰¹Pb, ²⁰²mPb, ²⁰³Pb, ²⁰⁹Pb, ²¹²Pb,¹⁰⁰Pd, ¹⁰¹Pd, ¹⁰³Pd, ¹⁰⁹Pd, ¹¹¹mPd, ¹¹²Pd, ¹⁴³Pm, ¹⁴⁸Pm, ¹⁴⁸mPm, ¹⁴⁹Pm,¹⁵¹Pm, ²⁰⁴Po, ²⁰⁶Po, ²⁰⁷Po, ²¹⁰Po, ¹³⁹Pr, ¹⁴²Pr, ¹⁴³Pr ¹⁴⁵Pr, ¹⁸⁸Pt,¹⁸⁹Pt, ¹⁹¹Pt, ¹⁹³mPt, ¹⁹⁵mPt, ¹⁹⁷Pt, ²⁰⁰Pt, ²⁰²Pt, ²³⁴Pu, ²³⁷Pu, ²⁴³Pu,²⁴⁵Pu, ²⁴⁶Pu, ²⁴⁷Pu, ²²³Ra, ²²⁴Ra, ²²⁵Ra, ⁸¹Rb, ⁸²Rb, ⁸²mRb, ⁸³Rb, ⁸⁴Rb,⁸⁶Rb, ¹⁸¹Re, ¹⁸²Re, ¹⁸²mRe, ¹⁸³Re, ¹⁸⁴Re, ¹⁸⁴mRe, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re,¹⁹⁰mRe, ⁹⁹Rh, ⁹⁹mRh, ¹⁰⁰Rh, ¹⁰¹mRh, ¹⁰²Rh, ¹⁰³mRh, ¹⁰⁵Rh, ²¹¹Rn, ²²²Rn,⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru, ³⁵S, ¹¹⁸mSb, ¹¹⁹Sb, ¹²⁰Sb, ¹²⁰mSb, ¹²²Sb, ¹²⁴Sb,¹²⁶Sb, ¹²⁷Sb, ¹²⁸Sb, ¹²⁹Sb, ⁴³Sc, ⁴⁴Sc, ⁴⁴mSc, ⁴⁶Sc, ⁴⁷Sc, ⁴⁸Sc, ⁷²Se,⁷³Se, ⁷⁵Se, ¹⁵³Sm, ¹⁵⁶Sm, ¹¹⁰Sn, ¹¹³Sn, ¹¹⁷mSn, ¹¹⁹mSn, ¹²¹Sn, ¹²³Sn,¹²⁵Sn, ⁸²Sr, ⁸³Sr, ⁸⁵Sr, ⁸⁹Sr, ⁹¹Sr, ¹⁷³Ta, ¹⁷⁵Ta, ¹⁷⁶Ta, ¹⁷⁷Ta, ¹⁸⁰Ta,¹⁸²Ta, ¹⁸³Ta, ¹⁸⁴Ta, ¹⁴⁹Tb, ¹⁵⁰Tb, ¹⁵¹Tb, ¹⁵²Tb, ¹⁵³Tb, ¹⁵⁴Tb, ¹⁵⁴mTb,¹⁵⁴m²Tb, ¹⁵⁵Tb, ¹⁵⁶Tb, ¹⁵⁶mTb, ¹⁵⁶m²Tb, ¹⁶⁰Tb, ¹⁶¹Tb, ⁹⁴Tc, ⁹⁵Tc, ⁹⁵mTc,⁹⁶Tc, ⁹⁷mTc, ⁹⁹mTc, ¹¹⁸Te, ¹¹⁹Te, ¹¹⁹mTe, ¹²¹Te, ¹²¹mTe, ¹²³mTe, ¹²⁵mTe,¹²⁷Te, ¹²⁷mTe, ¹²⁹mTe, ¹³¹mTe, ¹³²Te ²²⁷Th, ²³¹Th, ²³⁴Th, ⁴⁵Ti, ¹⁹⁸Tl,¹⁹⁹Tl, ²⁰⁰Tl, ²⁰¹Tl, ²⁰²Tl, ²⁰⁴Tl, ¹⁶⁵Tm, ¹⁶⁶Tm, ¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁷⁰Tm,¹⁷²Tm, ¹⁷³Tm, ²³⁰U, ²³¹U, ²³⁷U, ²⁴⁰U, ⁴⁸V, ¹⁷⁸W, ¹⁸¹W, ¹⁸⁵W, ¹⁸⁷W, ¹⁸⁸W,¹²²Xe, ¹²⁵Xe, ¹²⁷Xe, ¹²⁹mXe, ¹³¹mXe, ¹³³Xe, ¹³³mXe, ¹³⁵Xe, ⁸⁵mY, ⁸⁶Y,⁸⁷Y, ⁸⁷mY, ⁸⁸Y, ⁹⁰Y, ⁹⁰mY, ⁹¹Y, ⁹²Y, ⁹³Y, ¹⁶⁶Yb, ¹⁶⁹Yb, ¹⁷⁵Yb, ⁶²Zn,⁶⁵Zn, ⁶⁹mZn, ⁷¹mZn, ⁷²Zn, ⁸⁶Zr, ⁸⁸Zr, ⁸⁹Zr, ⁹⁵Zr, and ⁹⁷Zr.

It can be helpful to classify cytotoxic radionuclides into groups, forexample, metals (e.g., ⁹⁰Y, ⁶⁷Cu, ²¹³Bi, ²¹²Bi), and transitionalelements (e.g., ¹⁸⁶Re). Further, examples of pure β-emitters include⁶⁷Cu and ⁹⁰Y; and examples of α-emitters include ²¹³Bi. μ-emitters thatemit γ-radiation include ¹⁷⁷Lu and ¹⁸⁶Re, while Auger emitters andradionuclides that decay by internal conversion include ⁶⁷Ga.

In particular embodiments, SCCs can be charged with radionuclides thatare useful for imaging methods, such as PET imaging. An example of aradionuclide that is useful for PET imaging includes ⁸⁹Zr. ⁸⁹Zr has ahalf-life of 3 days and forms the daughter isotope ⁸⁹Y.

In particular embodiments, SCCs can be charged with radionuclides thatare useful for radiation therapy. Examples of radionuclides that areuseful for radiation therapy include ²²⁵Ac and ²²⁷Th. ²²⁵Ac is aradionuclide with the half-life of ten days. As ²²⁵Ac decays thedaughter isotopes ²²¹Fr, ²¹³Bi, and ²⁰⁹Pb are formed. ²²⁷Th has ahalf-life of 19 days, and forms the daughter isotope ²²³Ra.

As indicated, radionuclides can decay to form daughter isotopes. Inparticular embodiments, the SCCs disclosed herein retain daughterisotopes. For example, when an SCC is charged with ²²⁷Th, ²²³Ra daughterisotopes can be produced. In these embodiments, the ²²³Ra daughterisotope can be retained in the SCC longer than in classical chelatingconstructs due to encapsulation within the protein calyx. As isunderstood by one of ordinary skill in the art, such longer retentionmay be characterized by kinetic assays.

As will be appreciated by one of ordinary skill in the art, more thanone radioisotope may be chosen and used in particular nuclear medicineindications. Thus, embodiments can include a single species ofradioisotope, two species of radioisotopes, or a population of aplurality of species of radioisotopes combined in various proportions.In this manner the useful properties of different radioisotopes can becombined. For example, a single radioisotope decays at a linear rate. Bycombining radioisotopes of different half-lives, it is possible tocreate a non-linear decay rate.

Targeting Domains. In particular embodiments, SCCs disclosed hereininclude a targeting domain. Targeting domains can direct charged SCCs toimaging or therapeutic areas of interest. In particular embodiments, thetargeting domains direct the SCC to a region of the body that will beimaged using nuclear medicine diagnostic techniques. In particularembodiments, the targeting domains direct the SCC to a cell type that istargeted for radiotherapy.

In particular embodiments, targeting domains can be derived from wholeproteins or protein fragments with an affinity for particular tissuesand/or cell types of interest. In particular embodiments, targetingdomains can be derived from whole antibodies or binding fragments of anantibody, e.g., Fv, Fab, Fab′, F(ab′)₂, Fc, and single chain Fvfragments (scFvs) or any biologically effective fragments of animmunoglobulin that bind specifically to, for example, a cancer antigenepitope. Antibodies or antigen binding fragments include all or aportion of polyclonal antibodies, monoclonal antibodies, humanantibodies, humanized antibodies, synthetic antibodies, chimericantibodies, bispecific antibodies, mini bodies, and linear antibodies.

Targeting domains from human origin or humanized antibodies have loweredor no immunogenicity in humans and have a lower number ofnon-immunogenic epitopes compared to non-human antibodies. Antibodiesand their fragments will generally be selected to have a reduced levelor no antigenicity in human subjects. Targeting domains can particularlyinclude any peptide that specifically binds a selected unwanted cellepitope. Sources of targeting domains include antibody variable regionsfrom various species (which can be in the form of antibodies, sFvs,scFvs, Fabs, scFv-based grababody, or soluble VH domain or domainantibodies). These antibodies can form antigen-binding regions usingonly a heavy chain variable region, i.e., these functional antibodiesare homodimers of heavy chains only (referred to as “heavy chainantibodies”) (Jespers et al., Nat. Biotechnol. 22:1161, 2004;Cortez-Retamozo et al., Cancer Res. 64:2853, 2004; Baral et al., NatureMed. 12:580, 2006; and Barthelemy et al., J. Biol. Chem. 283:3639,2008).

Phage display libraries of partially or fully synthetic antibodies areavailable and can be screened for an antibody or fragment thereof thatcan bind a selected epitope. For example, targeting domains may beidentified by screening a Fab phage library for Fab fragments thatspecifically bind to a target of interest (see Hoet et al., Nat.Biotechnol. 23:344, 2005). Phage display libraries of human antibodiesare also available. Additionally, traditional strategies for hybridomadevelopment using a target of interest as an immunogen in convenientsystems (e.g., mice, HuMAb Mouse®, TC Mouse™, KM-Mouse®, llamas,chicken, rats, hamsters, rabbits, etc.) can be used to develop targetingdomains. In particular embodiments, antibodies specifically bind toselected epitopes expressed by targeted cells and do not cross reactwith nonspecific components or unrelated targets. Once identified, theamino acid sequence or polynucleotide sequence coding for the antibodycan be isolated and/or determined.

An alternative source of targeting domains includes sequences thatencode random peptide libraries or sequences that encode an engineereddiversity of amino acids in loop regions of alternative non-antibodyscaffolds, such as scTCR (see, e.g., Lake et al., Int. Immunol. 11:745,1999; Maynard et al., J. Immunol. Methods 306:51, 2005; U.S. Pat. No.8,361,794), mAb² or Fcab™ (see, e.g., PCT Patent Application PublicationNos. WO 2007/098934; WO 2006/072620), affibody, avimers, fynomers,cytotoxic T-lymphocyte associated protein-4 (Weidle et al., Cancer Gen.Proteo. 10:155, 2013), or the like (Nord et al., Protein Eng. 8:601,1995; Nord et al., Nat. Biotechnol. 15:772, 1997; Nord et al., Euro. J.Biochem. 268:4269, 2001; Binz et al., Nat. Biotechnol. 23:1257, 2005;Boersma and Plückthun, Curr. Opin. Biotechnol. 22:849, 2011).

In particular embodiments, an antibody fragment is used as the targetingdomain of a SCC. An “antibody fragment” denotes a portion of a completeor full length antibody that retains the ability to bind to an epitope.Examples of antibody fragments include Fv, scFv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; and linear antibodies.

A single chain variable fragment (scFv) is a fusion protein of thevariable regions of the heavy and light chains of immunoglobulinsconnected with a short linker peptide. Fv fragments include the VL andVH domains of a single arm of an antibody. Although the two domains ofthe Fv fragment, VL and VH, are coded by separate genes, they can bejoined, using, for example, recombinant methods, by a synthetic linkerthat enables them to be made as a single protein chain in which the VLand VH regions pair to form monovalent molecules (single chain Fv(scFv)). For additional information regarding Fv and scFv, see e.g.,Bird, et al., Science 242 (1988) 423-426; Huston, et al., Proc. Natl.Acad. Sci. USA 85 (1988) 5879-5883; Plueckthun, in The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore (eds.),Springer-Verlag, New York), (1994) 269-315; WO1993/16185; U.S. Pat. Nos.5,571,894; and 5,587,458.

A Fab fragment is a monovalent antibody fragment including VL, VH, CLand CH1 domains. A F(ab′)₂ fragment is a bivalent fragment including twoFab fragments linked by a disulfide bridge at the hinge region. Fordiscussion of Fab and F(ab′)₂ fragments having increased in vivohalf-life, see U.S. Pat. No. 5,869,046. Diabodies include twoepitope-binding sites that may be bivalent. See, for example, EP0404097; WO1993/01161; and Holliger, et al., Proc. Natl. Acad. Sci. USA90 (1993) 6444-6448. Dual affinity retargeting antibodies (DART™; basedon the diabody format but featuring a C-terminal disulfide bridge foradditional stabilization (Moore et al., Blood 117, 4542-51 (2011))) canalso be used. Antibody fragments can also include isolated CDRs. For areview of antibody fragments, see Hudson, et al., Nat. Med. 9 (2003)129-134.

Antibody fragments can be made by various techniques, includingproteolytic digestion of an intact antibody as well as production byrecombinant host-cells (e.g. E. coli or phage), as described herein.Antibody fragments can be screened for their binding properties in thesame manner as intact antibodies.

In particular embodiments, targeting domains can also include a naturalreceptor or ligand for an epitope. For example, if a target for bindingincludes PD-L1, the binding domain can include PD-1 (including, e.g., aPD-1/antiCD3 fusion). One example of a receptor fusion for binding isEnbrel® (Immunex). Natural receptors or ligands can also be modified toenhance binding. For example, betalacept is a modified version ofabatacept.

Binding can also be enhanced through increasing avidity. Any screeningmethod known in the art can be used to identify increased avidity to anantigen epitope.

As used herein, an epitope denotes the binding site on a protein targetbound by a corresponding targeting domain. The targeting domain eitherbinds to a linear epitope, (e.g., an epitope including a stretch of 5 to12 consecutive amino acids), or the targeting domains binds to athree-dimensional structure formed by the spatial arrangement of severalshort stretches of the protein target. Three-dimensional epitopesrecognized by a targeting domain, e.g. by the epitope recognition siteor paratope of an antibody or antibody fragment, can be thought of asthree-dimensional surface features of an epitope molecule. Thesefeatures fit precisely (in) to the corresponding binding site of thetargeting domains and thereby binding between the targeting domains andits target protein is facilitated.

“Bind” means that the targeting domain associates with its targetepitope with a dissociation constant (1(D) of 10⁻⁸ M or less, in oneembodiment of from 10⁻⁵ M to 10⁻¹³ M, in one embodiment of from 10⁻⁵ Mto 10⁻¹⁰ M, in one embodiment of from 10⁻⁵ M to 10⁻⁷ M, in oneembodiment of from 10⁻⁸ M to 10⁻¹³ M, or in one embodiment of from 10⁻⁹M to 10⁻¹³ M. The term can be further used to indicate that thetargeting domains does not bind to other biomolecules present, (e.g., itbinds to other biomolecules with a dissociation constant (KD) of 10⁻⁴ Mor more, in one embodiment of from 10⁻⁴ M to 1 M.

In particular embodiments, targeting domains of SCCs can be designed totarget cancer cell antigens. Cancer cell antigens are preferentiallyexpressed by cancer cells. “Preferentially expressed” means that acancer cell antigen is found at higher levels on cancer cells ascompared to other cell types. The difference in expression level issignificant enough that, within sound medical judgment, administrationof therapeutics selectively targeting the cancer cells based on thepresence of the cancer antigen outweighs the risk of collateral killingof other non-cancer cells that may also express the marker to a lesserdegree. In some instances, a cancer antigen is only expressed by thetargeted cancer cell type. In other instances, the cancer antigen isexpressed on the targeted cancer cell type at least 25%, 35%, 45%, 55%,65%, 75%, 85%, 95%, 96%, 97%, 98%, 99%, or 100% more than onnon-targeted cells.

The following table provides examples of particular cancer antigens thatcan be targeted by SCCs.

Targeted Cancer Cancer Antigens Leukemia/Lymphoma CD19, CD20, CD22,ROR1, CD33, WT-1 Multiple Myeloma B-cell maturation antigen (BCMA)Prostate Cancer PSMA, WT1, Prostate Stem Cell antigen (PSCA), SV40 TBreast Cancer HER2, ERBB2, ROR1 Stem Cell Cancer CD133 Ovarian CancerL1-CAM, extracellular domain of MUC16 (MUC-CD), folate binding protein(folate receptor), Lewis Y, ROR1, mesothelin, WT-1 Mesotheliomamesothelin Renal Cell Carcinoma carboxy-anhydrase-IX (CAIX); MelanomaGD2 Pancreatic Cancer mesothelin, CEA, CD24, ROR1 Lung Cancer ROR1

In more particular examples, cancer cell antigens include:

Cancer Antigen Sequence PSMA MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVD PSKAWGEVKRQIYVAAFTVQAAAETLSEVA(SEQ ID NO: 26) PSCA MKAVLLALLMAGLALQPGTALLCYSCKAQVSNEDCLQVENCTQLGEQCWTARIRAVGLLTVISKGCSLNCVDDSQDYYVGKKNITCCDTDLCNASGAHALQPAAAILA LLPALGLLLWGPGQL (SEQ ID NO: 27)Mesothelin MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDGVLANPPNISSLSPRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQLRCLAHRLSEPPEDLDALPLDLLLFLNPDAFSGPQACTHFFSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLLSEADVRALGGLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTWSVSTMDALRGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKTACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSVQEALSGTPCLLGPGPVLTV LALLLASTLA (SEQ ID NO: 28) CD19MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLASWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCVPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQRALVLRRKRKRMTDPTRRFFKVTPPPGSGPQNQYGNVLSLPTPTSGLGRAQRWAAGLGGTAPSYGNPSSDVQADGALGSRSPPGVGPEEEEGEGYEEPDSEEDSEFYENDSNLGQDQLSQDGSGYENPEDEPLGPEDEDSFSNAESYENEDEELTQPVARTMDFLSPHGSAWDPSREATSLGSQSYEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENMDNPDGP DPAWGGGGRMGTWSTR (SEQ ID NO: 29)CD20 MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKTLGAVQIMNGLFHIALGGLLMIPAGIYAPICVTVWYPLWGGIMYIISGSLLAATEKNSRKCLVKGKMIMNSLSLFAAISGMILSIMDILNIKISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQSLFLGILSVMLIFAFFQELVIAGIVENEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQES SPIENDSSP (SEQ ID NO: 30) ROR1MHRPRRRGTRPPLLALLAALLLAARGAAAQETELSVSAELVPTSSWNISSELNKDSYLTLDEPMNNITTSLGQTAELHCKVSGNPPPTIRWFKNDAPVVQEPRRLSFRSTIYGSRLRIRNLDTTDTGYFQCVATNGKEVVSSTGVLFVKFGPPPTASPGYSDEYEEDGFCQPYRGIACARFIGNRTVYMESLHMQGEIENQITAAFTMIGTSSHLSDKCSQFAIPSLCHYAFPYCDETSSVPKPRDLCRDECEILENVLCQTEYIFARSNPMILMRLKLPNCEDLPQPESPEAANCIRIGIPMADPINKNHKCYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFPELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPACDSKDSKEKNKMEILYILVPSVAIPLAIALLFFFICVCRNNQKSSSAPVQRQPKHVRGQNVEMSMLNAYKPKSKAKELPLSAVRFMEELGECAFGKIYKGHLYLPGMDHAQLVAIKTLKDYNNPQQWTEFQQEASLMAELHHPNIVCLLGAVTQEQPVCMLFEYINQGDLHEFLIMRSPHSDVGCSSDEDGTVKSSLDHGDFLHIAIQIAAGMEYLSSHFFVHKDLAARNILIGEQLHVKISDLGLSREIYSADYYRVQSKSLLPIRWMPPEAIMYGKFSSDSDIWSFGVVLWEIFSFGLQPYYGFSNQEVIEMVRKRQLLPCSEDCPPRMYSLMTECWNEIPSRRPRFKDIHVRLRSWEGLSSHTSSTTPSGGNATTQTTSLSASPVSNLSNPRYPNYMFPSQGITPQGQIAGFIGPPIPQNQRFIPINGYPIPPGYAAFPAAHYQPTGPPRVIQHCPPPKSRSPSSASGSTSTGHVTSLPSSGSNQEANIPLLPHMSIPNHPGGMGITVFGNKSQKPYKIDSKQASLLGDANIHGHTESMISAE L (SEQ ID NO: 31) WT1MGHHHHHHHHHHSSGHIEGRHMRRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFFRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFSCRWPSCQKKF ARSDELVRHHNMHQRNMTKLQLAL(SEQ ID NO: 32)

In particular embodiments, the targeting domains of the SCC targets aRORI epitope. In particular embodiments, the targeting domains of theSCC is a human or humanized scFv including a variable light chainincluding a CDRL1 sequence of ASGFDFSAYYM (SEQ ID NO: 33), CDRL2sequence of TIYPSSG (SEQ ID NO: 34), and a CDRL3 sequence of ADRATYFCA(SEQ ID NO: 35). In particular embodiments, the targeting domain of theSCC is a human or humanized scFv including a variable heavy chainincluding CDRH1 sequence of DTIDWY (SEQ ID NO: 36), CDRH2 sequence ofVQSDGSYTKRPGVPDR (SEQ ID NO: 37), and a CDRH3 sequence of YIGGYVFG (SEQID NO: 38). A number of antibodies specific for RORI are known to thoseof skill in the art and can be readily characterized for sequence,epitope binding, and affinity.

In a particular embodiment, the targeting domain of the SCC binds to aCD19 epitope. In particular embodiments, the targeting domain of the SCCis a single chain Fv fragment (scFv) that includes VH and VL regionsspecific for CD19. In certain embodiments, the V_(H) and V_(L) regionsare human. Exemplary V_(H) and V_(L) regions include the segments ofanti-CD19 specific monoclonal antibody FMC63. In particular embodiments,the scFV is a human or humanized and includes a variable light chainincluding a CDRL1 sequence of RASQDISKYLN (SEQ ID NO: 39), CDRL2sequence of SRLHSGV (SEQ ID NO: 40), and a CDRL3 sequence of GNTLPYTFG(SEQ ID NO: 41). In other embodiments, the scFV is a human or humanizedScFv including a variable heavy chain including CDRH1 sequence of DYGVS(SEQ ID NO: 42), CDRH2 sequence of VTWGSETTYYNSALKS (SEQ ID NO: 43), anda CDRH3 sequence of YAMDYWG (SEQ ID NO: 44). Other CD19-targetingantibodies such as SJ25C1 and HD37 are known. (SJ25C1: Bejcek et al.Cancer Res 2005, PMID 7538901; HD37: Pezutto et al. JI 1987, PMID2437199).

In particular embodiments, the targeting domain of the SCC targets aPSMA epitope. A number of antibodies specific for PSMA are known tothose of skill in the art and can be readily characterized for sequence,epitope binding, and affinity. Targeting domains can also includeanti-Mesothelin ligands (associated with treating ovarian cancer,pancreatic cancer, and mesothelioma). As will be understood by one ofordinary skill in the art, the targeting domains can bind any number ofepitopes on the cancer antigens disclosed herein (among others).

Rituxan (Rituximab, Genentech) targets CD20 for CD20-positivenon-Hodgkin's lymphoma and Arzerra (Ofatumumab, Novartis), targets adifferent epitope of CD20. Herceptin can also be used.

Proteins disclosed herein include variants. Variants of proteinsdisclosed herein include proteins having one or more amino acidadditions, deletions, stop positions, or substitutions, as compared to aprotein disclosed herein. To qualify as a variant, the altered proteinmust provide an equivalent or improved intended effect as compared to areference protein provided elsewhere herein. Equivalent means notstatistically significantly different. Improved means higher affinitybinding.

An amino acid substitution can be a conservative or a non-conservativesubstitution. Variants of proteins disclosed herein can include thosehaving one or more conservative amino acid substitutions. A conservativesubstitution involves a substitution found in one of the followingconservative substitutions groups: Group 1: alanine (Ala or A), glycine(Gly or G), Ser, Thr; Group 2: aspartic acid (Asp or D), E; Group 3:asparagine (Asn or N), glutamine (Gln or Q); Group 4: Arg, lysine (Lysor K), histidine (His or H); Group 5: Ile, leucine (Leu or L),methionine (Met or M), valine (Val or V); and Group 6: F, Tyr, W.

Additionally, amino acids can be grouped into conservative substitutiongroups by similar function, chemical structure, or composition (e.g.,acidic, basic, aliphatic, aromatic, sulfur-containing). For example, analiphatic grouping may include, for purposes of substitution, G, A, V,L, and I. Other groups containing amino acids that are consideredconservative substitutions for one another include: sulfur-containing: Mand C; acidic: D, E, N, and Q; small aliphatic, nonpolar or slightlypolar residues: A, S, T, P, and G; polar, negatively charged residuesand their amides: D, N, E, and Q; polar, positively charged residues: H,R, and K; large aliphatic, nonpolar residues: M, L, I, V, and C; andlarge aromatic residues: F, Y, and W. Additional information is found inCreighton (1984) Proteins, W.H. Freeman and Company.

Variants of proteins disclosed herein also include sequences with atleast 70% sequence identity, at least 80% sequence identity, at least85% sequence, at least 90% sequence identity, at least 95% sequenceidentity, at least 96% sequence identity, at least 97% sequenceidentity, at least 98% sequence identity, or at least 99% sequenceidentity to a protein disclosed herein. More particularly, variants ofthe proteins disclosed herein include proteins that share: 70% sequenceidentity with any of e.g., SEQ ID NO: 1-46; 80% sequence identity withany of e.g., SEQ ID NO: 1-46; 81% sequence identity with any of e.g.,SEQ ID NO: 1-46; 82% sequence identity with any of e.g., SEQ ID NO:1-46; 83% sequence identity with any of e.g., SEQ ID NO: 1-46; 84%sequence identity with any of e.g., SEQ ID NO: 1-46; 85% sequenceidentity with any of e.g., SEQ ID NO: 1-46; 86% sequence identity withany of e.g., SEQ ID NO: 1-46; 87% sequence identity with any of e.g.,SEQ ID NO: 1-46; 88% sequence identity with any of e.g., SEQ ID NO:1-46; 89% sequence identity with any of e.g., SEQ ID NO: 1-46; 90%sequence identity with any of e.g., SEQ ID NO: 1-1-46; 91% sequenceidentity with any of e.g., SEQ ID NO: 1-46; 92% sequence identity withany of e.g., SEQ ID NO: 1-46; 93% sequence identity with any of e.g.,SEQ ID NO: 1-46; 94% sequence identity with any of e.g., SEQ ID NO:1-46; 95% sequence identity with any of e.g., SEQ ID NO: 1-46; 96%sequence identity with any of e.g., SEQ ID NO: 1-46; 97% sequenceidentity with any of e.g., SEQ ID NO: 1-46; 98% sequence identity withany of e.g., SEQ ID NO: 1-46; or 99% sequence identity with any of e.g.,SEQ ID NO: 1-46.

% sequence identity refers to a relationship between two or moresequences, as determined by comparing the sequences. In the art,identity also means the degree of sequence relatedness between sequencesas determined by the match between strings of such sequences. Identity(often referred to as similarity) can be readily calculated by knownmethods, including those described in: Computational Molecular Biology(Lesk, A. M., ed.) Oxford University Press, N Y (1988); Biocomputing:Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, N Y(1994); Computer Analysis of Sequence Data, Part I (Griffin, A. M., andGriffin, H. G., eds.) Humana Press, N J (1994); Sequence Analysis inMolecular Biology (Von Heijne, G., ed.) Academic Press (1987); andSequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) OxfordUniversity Press, NY (1992). Preferred methods to determine sequenceidentity are designed to give the best match between the sequencestested. Methods to determine sequence identity and similarity arecodified in publicly available computer programs. Sequence alignmentsand percent identity calculations may be performed using the Megalignprogram of the LASERGENE bioinformatics computing suite (DNASTAR, Inc.,Madison, Wis.). Multiple alignment of the sequences can also beperformed using the Clustal method of alignment (Higgins and SharpCABIOS, 5, 151-153 (1989) with default parameters (GAP PENALTY=10, GAPLENGTH PENALTY=10). Relevant programs also include the GCG suite ofprograms (Wisconsin Package Version 9.0, Genetics Computer Group (GCG),Madison, Wis.); BLASTP, BLASTN, BLASTX (Altschul, et al., J. Mol. Biol.215:403-410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wis.); and theFASTA program incorporating the Smith-Waterman algorithm (Pearson,Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date1992, 111-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York,N.Y. Within the context of this disclosure it will be understood thatwhere sequence analysis software is used for analysis, the results ofthe analysis are based on the default values of the program referenced.Default values mean any set of values or parameters which originallyload with the software when first initialized.

D-substituted analogs include protein disclosed herein having one moreL-amino acids substituted with one or more D-amino acids. The D-aminoacid can be the same amino acid type as that found in the referencesequence or can be a different amino acid. Accordingly, D-analogs canalso be variants.

While exemplary sequences are provided herein, sequence informationprovided by public databases can be used to identify additional relatedand relevant protein sequences and associated nucleic acid sequencesencoding such proteins.

Methods of Protein Production. Embodiments disclosed herein utilizesiderocalin (Scn), in particular embodiments, in combination or as afusion with a targeting domain. In particular embodiments, proteinsdisclosed herein are formed using the Daedalus expression system asdescribed in Pechman et al., Am J Physiol 294: R1234-R1239, 2008. TheDaedalus system utilizes inclusion of minimized ubiquitous chromatinopening elements in transduction vectors to reduce or prevent genomicsilencing and to help maintain the stability of decigram levels ofexpression. This system can bypass tedious and time-consuming steps ofother protein production methods by employing the secretion pathway ofserum-free adapted human suspension cell lines, such as 293 Freestyle.Using optimized lentiviral vectors, yields of 20-100 mg/I of correctlyfolded and post-translationally modified, endotoxin-free protein of upto 70 kDa in size, can be achieved in conventional, small-scale (100 ml)culture.

In particular embodiments, the amount of peptide obtained can be between10 mg/L and 200 mg/L, between 50 mg/L and 200 mg/L, between 100 mg/L and200 mg/L, and between 150 mg/L and 200 mg/L. At these yields, mostproteins can be purified using a single size-exclusion chromatographystep, immediately appropriate for use in structural, biophysical ortherapeutic applications. Bandaranayake et al., Nucleic Acids Res., 2011(November); 39(21). In some instances, purification by chromatographymay not be needed due to the purity of manufacture according the methodsdescribed herein. Further, Scn when loaded with siderophores and iron,has a deep red color that can aid in chromatography or otherpurification steps.

Particular embodiments utilize DNA constructs (e.g., chimeric genes,expression cassettes, expression vectors, recombination vectors, etc.)including a nucleic acid sequence encoding the protein or proteins ofinterest operatively linked to appropriate regulatory sequences. SuchDNA constructs are not naturally-occurring DNA molecules and are usefulfor introducing DNA into host-cells to express selected proteins ofinterest.

Operatively linked refers to the linking of DNA sequences (including theorder of the sequences, the orientation of the sequences, and therelative spacing of the various sequences) in such a manner that theencoded protein is expressed. Methods of operatively linking expressioncontrol sequences to coding sequences are well known in the art. See,e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor, N. Y., 1982; and Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor, N. Y., 1989.

Expression control sequences are DNA sequences involved in any way inthe control of transcription or translation. Suitable expression controlsequences and methods of making and using them are well known in theart. Expression control sequences generally include a promoter. Thepromoter may be inducible or constitutive. It may benaturally-occurring, may be composed of portions of variousnaturally-occurring promoters, or may be partially or totally synthetic.Guidance for the design of promoters is provided by studies of promoterstructure, such as that of Harley and Reynolds, Nucleic Acids Res., 15,2343-2361, 1987. Also, the location of the promoter relative to thetranscription start may be optimized. See, e.g., Roberts et al., Proc.Natl. Acad. Sci. USA, 76:760-764, 1979.

The promoter may include, or be modified to include, one or moreenhancer elements. In particular embodiments, the promoter will includea plurality of enhancer elements. Promoters containing enhancer elementscan provide for higher levels of transcription as compared to promotersthat do not include them.

For efficient expression, the coding sequences can be operatively linkedto a 3′ untranslated sequence. In particular embodiments, the 3′untranslated sequence can include a transcription termination sequenceand a polyadenylation sequence. The 3′ untranslated region can beobtained, for example, from the flanking regions of genes.

In particular embodiments, a 5′ untranslated leader sequence can also beemployed. The 5′ untranslated leader sequence is the portion of an mRNAthat extends from the 5′ CAP site to the translation initiation codon.

In particular embodiments, a “hisavi” tag can be added to the N-terminusor C-terminus of a gene by the addition of nucleotides coding for theAvitag amino acid sequence, GLNDIFEAQKIEWHE (SEQ ID NO: 45), as well asthe 6×histidine tag coding sequence, HHHHHH (SEQ ID NO: 46). The Avitagavidity tag can be biotinylated by a biotin ligase to allow forbiotin-avidin or biotin-streptavidin based interactions for proteinpurification, as well as for immunobiology (such as immunoblotting orimmunofluorescence) using anti-biotin antibodies. The 6×histidine tagallows for protein purification using Ni-2+ affinity chromatography.

In particular embodiments, expressed fusion proteins can include or beencoded by an IgK starter sequence, a sFLAG, a HIS, and a TEV. Incertain embodiments, the fusion protein includes the followingconstruct: IgK SP-sFLAG-HIS-siderocalin-TEV-peptide. In someembodiments, the fusion protein is generated by direct fusion of eachsubunit to the adjacent subunits. In certain embodiments, thecomposition further includes a linker sequence between the targetingdomain and the Scn protein.

Nucleic acid sequences encoding proteins disclosed herein can be derivedby those of ordinary skill in the art. Nucleic acid sequences can alsoinclude one or more of various sequence polymorphisms, mutations, and/orsequence variants. In particular embodiments, the sequencepolymorphisms, mutations, and/or sequence variants do not affect thefunction of the encoded protein. The sequences can also includedegenerate codons of a native sequence or sequences that may beintroduced to provide codon preference.

In some aspects, the DNA constructs can be introduced by transfection, atechnique that involves introduction of foreign DNA into the nucleus ofeukaryotic cells. In some aspects, the proteins can be synthesized bytransient transfection (DNA does not integrate with the genome of theeukaryotic cells, but the genes are expressed for 24-96 hours). Variousmethods can be used to introduce the foreign DNA into the host-cells,and transfection can be achieved by chemical-based means including bythe calcium phosphate, by dendrimers, by liposomes, and by the use ofcationic polymers. Non-chemical methods of transfection includeelectroporation, sono-poration, optical transfection, protoplast fusion,impalefection, and hydrodynamic delivery. In some embodiments,transfection can be achieved by particle-based methods including genegun where the DNA construct is coupled to a nanoparticle of an inertsolid which is then “shot” directly into the target-cell's nucleus.Other particle-based transfection methods include magnet assistedtransfection and impalefection.

Methods of Synthesizing Chelators. In some embodiments, compositions ofchelators described herein can be synthesized using techniques that aresimpler and less harsh than conventional techniques. In particular, theuse of dichlorophenylmethane improves the synthesis of naturalsiderophores and analogs, such as 3,4,3-LI(CAM), by minimizing the useof harsh, toxic substances in the synthesis of siderophores andsiderophore-like ligands. Additionally, the reaction conditions areimproved when dichlorophenylmethane is used in the synthesis ofsiderophores and siderophore-like ligands.

Methods of Making Siderocalin-Chelator Combinations. In particularembodiments, SCCs can be made by contacting Scn or Scn-targeting domainfusion proteins with chelators and allowing complexes between the twomolecules to form.

Methods of Making Radionuclides. Radioisotopes can be obtained insolution in water or other polar fluid in elemental form (i.e.,uncharged) or ionic form. As appreciated by the skilled artisan, when inionic form, radioisotopes may occur in various different valence states,as anions, or as cations, depending upon the particular radioisotopebeing considered.

Methods of Charging Chelators with Radionuclides. In particularembodiments, chelators can be charged with radionuclides by contactingthe chelators with metallic radioisotopes and allowing complexes betweenthe two molecules to form.

Methods of Making Siderocalin-Chelator-Radionuclide Complexes. Inparticular embodiments, SCC-metal complexes can be made by contactingSCCs with metallic radioisotopes and allowing complexes between themolecules to form. In other embodiments, SCC-metal complexes can be madeby contacting chelator-metal combinations with siderocalins and allowingcomplexes between the molecules to form.

Formulations. The various forms of Scn-chelator combinations (SCCs) andcharged SCCs described herein, are referred to herein as activeingredients. Active ingredients also include prodrugs, salts, analogs,and/or derivatives of SCCs, charged SCCs, or portions of SCCs or chargedSCCs.

A prodrug includes an active ingredient which is converted into atherapeutically active or more therapeutically active compound afteradministration, such as by cleavage of a protein.

A pharmaceutically acceptable salt includes any salt that retains theactivity of the active ingredient and is acceptable for pharmaceuticaluse. A pharmaceutically acceptable salt also refers to any salt whichmay form in vivo as a result of administration of an acid, another salt,or a prodrug which is converted into an acid or salt. Suitablepharmaceutically acceptable acid addition salts can be prepared from aninorganic acid or an organic acid. Examples of inorganic acids includehydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric andphosphoric acid. Appropriate organic acids can be selected fromaliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic,carboxylic and sulfonic classes of organic acids. Suitablepharmaceutically acceptable base addition salts include metallic saltsmade from aluminum, calcium, lithium, magnesium, potassium, sodium andzinc or organic salts made from N,N′-dibenzylethylene-diamine,chloroprocaine, choline, diethanolamine, ethylenediamine,N-methylglucamine, lysine, arginine and procaine.

The term analog (also structural analog or chemical analog) is used torefer to a compound that is structurally similar to another compound butdiffers with respect to a certain component, such as an atom, afunctional group, or a substructure. The term derivative refers to acompound that is obtained from a similar compound or a precursorcompound by a chemical reaction. As used herein, analogs and derivativesretain the therapeutic effectiveness of the parent compound (i.e., thereis no statistically significant difference in therapeutic activityaccording to an imaging assay or assessment of clinical improvement) orhave improved therapeutic effectiveness as defined elsewhere herein.

Active ingredients are formulated into compositions for administrationto subjects. Compositions include at least one active ingredient and atleast one pharmaceutically acceptable carrier. In particularembodiments, compositions include active ingredients of at least 0.1%w/v or w/w of the composition; at least 1% w/v or w/w of composition; atleast 10% w/v or w/w of composition; at least 20% w/v or w/w ofcomposition; at least 30% w/v or w/w of composition; at least 40% w/v orw/w of composition; at least 50% w/v or w/w of composition; at least 60%w/v or w/w of composition; at least 70% w/v or w/w of composition; atleast 80% w/v or w/w of composition; at least 90% w/v or w/w ofcomposition; at least 95% w/v or w/w of composition; or at least 99% w/vor w/w of composition.

Exemplary generally used pharmaceutically acceptable carriers includeany and all absorption delaying agents, antioxidants, binders, bufferingagents, bulking agents or fillers, chelating agents, coatings,disintegration agents, dispersion media, gels, isotonic agents,lubricants, preservatives, salts, solvents or co-solvents, stabilizers,surfactants, and/or delivery vehicles.

Exemplary antioxidants include ascorbic acid, methionine, and vitamin E.

Exemplary buffering agents include citrate buffers, succinate buffers,tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers,lactate buffers, acetate buffers, phosphate buffers, histidine buffers,and/or trimethylamine salts.

An exemplary chelating agent for use as a pharmaceutically acceptablecarrier is EDTA. Other chelating agents disclosed herein may also beused.

Exemplary isotonic agents include polyhydric sugar alcohols includingtrihydric or higher sugar alcohols, such as glycerin, erythritol,arabitol, xylitol, sorbitol, or mannitol.

Exemplary preservatives include phenol, benzyl alcohol, meta-cresol,methyl paraben, propyl paraben, octadecyldimethylbenzyl ammoniumchloride, benzalkonium halides, hexamethonium chloride, alkyl parabenssuch as methyl or propyl paraben, catechol, resorcinol, cyclohexanol,and 3-pentanol.

Stabilizers refer to a broad category of excipients which can range infunction from a bulking agent to an additive which solubilizes theactive ingredient or helps to prevent denaturation or adherence to thecontainer wall. Typical stabilizers can include polyhydric sugaralcohols; amino acids, such as R, K, G, Q, N, H, A, ornithine,L-leucine, 2-F, E, and T; organic sugars or sugar alcohols, such aslactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol,myoinisitol, galactitol, glycerol, and cyclitols, such as inositol; PEG;amino acid polymers; sulfur-containing reducing agents, such as urea,glutathione, thioctic acid, sodium thioglycolate, thioglycerol,alpha-monothioglycerol, and sodium thiosulfate; low molecular weightpolypeptides (i.e., <10 residues); proteins such as human serum albumin,bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidone; monosaccharides such as xylose, mannose,fructose and glucose; disaccharides such as lactose, maltose andsucrose; trisaccharides such as raffinose, and polysaccharides such asdextran. Stabilizers are typically present in the range of from 0.1 to10,000 parts by weight based on active ingredient weight.

In particular embodiments, the compositions disclosed herein can beformulated for administration by injection (e.g., intravenousinjection). Compositions can also be formulated for administration by,for example, inhalation, infusion, perfusion, lavage, or ingestion. Thecompositions disclosed herein can further be formulated for intradermal,intraarterial, intranodal, intralymphatic, intraperitoneal,intralesional, intraprostatic, intravaginal, intrarectal, topical,intrathecal, intravesicular, oral and/or subcutaneous administration andmore particularly by intravenous, intradermal, intraarterial,intranodal, intralymphatic, intraperitoneal, intralesional,intraprostatic, intravaginal, intrarectal, intrathecal, intramuscular,intravesicular, and/or subcutaneous injection.

For injection, compositions can be formulated as aqueous solutions, suchas in buffers including Hanks' solution, Ringer's solution, orphysiological saline. The aqueous solutions can contain formulatoryagents such as suspending, stabilizing, and/or dispersing agents.Alternatively, the formulation can be in lyophilized and/or powder formfor constitution with a suitable vehicle, e.g., sterile pyrogen-freewater, before use. Particular embodiments are formulated for intravenousor intramuscular administration.

For oral administration, the compositions can be formulated as tablets,pills, dragees, capsules, liquids, gels, syrups, slurries, suspensionsand the like. Compositions can be formulated as an aerosol forinhalation. In one embodiment, the aerosol is provided as part of ananhydrous, liquid or dry powder inhaler. Compositions can also beformulated as depot preparations. Depot preparations can be formulatedwith suitable polymeric or hydrophobic materials (for example as anemulsion in an acceptable oil) or ion exchange resins, or as sparinglysoluble derivatives, for example, as a sparingly soluble salts.Additionally, compositions can be formulated as sustained-releasesystems utilizing semipermeable matrices of solid polymers containing atleast one active ingredient.

Any composition disclosed herein can advantageously include any otherpharmaceutically acceptable carriers which include those that do notproduce significantly adverse, allergic, or other untoward reactionsthat outweigh the benefit of administration. Exemplary pharmaceuticallyacceptable carriers and formulations are disclosed in Remington'sPharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Moreover,formulations can be prepared to meet sterility, pyrogenicity, generalsafety, and purity standards as required by U.S. FDA Office ofBiological Standards and/or other relevant foreign regulatory agencies.

Kits. Also disclosed herein are kits including one or more containersincluding one or more of the active ingredients, compositions, Scnproteins, chelators, and/or radionuclides described herein. In variousembodiments, the kits may include one or more containers containing oneor more portions of active ingredients and/or compositions to be used incombination with other portions of the active ingredients and/orcompositions described herein. Associated with such container(s) can bea notice in the form prescribed by a governmental agency regulating themanufacture, use, or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use, orsale for human administration.

Optionally, the kits described herein further include instructions forusing the kit in the methods disclosed herein. In various embodiments,the kit may include instructions regarding preparation of the activeingredients and/or compositions for administration; administration ofthe active ingredients and/or compositions; appropriate reference levelsto interpret results associated with using the kit; proper disposal ofthe related waste; and the like. The instructions can be in the form ofprinted instructions provided within the kit or the instructions can beprinted on a portion of the kit itself. Instructions may be in the formof a sheet, pamphlet, brochure, CD-Rom, or computer-readable device, orcan provide directions to instructions at a remote location, such as awebsite. The instructions may be in English and/or in any national orregional language. In various embodiments, possible side effects andcontraindications to further use of components of the kit based on asubject's symptoms can be included.

In various embodiments, the kits described herein include some or all ofthe necessary medical supplies needed to use the kit effectively,thereby eliminating the need to locate and gather such medical supplies.Such medical supplies can include syringes, ampules, tubing, facemasks,protective clothing, a needleless fluid transfer device, an injectioncap, sponges, sterile adhesive strips, Chloraprep, gloves, and the like.Variations in contents of any of the kits described herein can be made.Particular kits provide materials to administer compositions throughintravenous administration.

Methods of Use. Methods disclosed herein include treating subjects(humans, veterinary animals (dogs, cats, reptiles, birds, etc.)livestock (horses, cattle, goats, pigs, chickens, etc.) and researchanimals (monkeys, rats, mice, fish, etc.) with therapeutic compositionsdisclosed herein. Treating subjects includes delivering therapeuticallyeffective amounts. Therapeutically effective amounts include those thatprovide effective amounts and therapeutic treatments.

An “effective amount” is the amount of a composition necessary to resultin a desired physiological change in the subject. Effective amounts areoften administered for research purposes. Effective amounts disclosedherein can cause a statistically-significant effect in an animal modelassessing a use of nuclear medicine.

A “therapeutic treatment” can include a treatment administered to asubject in need of imaging. The subject can be in need of imaging to aidin diagnosis; to locate a position for a therapeutic intervention; toassess the functioning of a body part; and/or to assess the presence orabsence of a condition. The effectiveness of a therapeutic imagingtreatment can be confirmed based on the capture of an image sufficientfor its intended purpose.

Exemplary types of imaging that utilize nuclear medicine include:positron emission tomography (PET), single photon emission computedtomography, radioisotope renography, and scintigraphy.

A “therapeutic treatment” can also include a treatment administered to asubject with a condition. The therapeutic treatment reduces, controls,or eliminates the condition or a symptom associated with the condition.Conditions treated with nuclear medicine include those associated withthe proliferation of unwanted cells.

In particular embodiments, therapeutic treatments reduce cellularproliferation. Cellular proliferation refers to the process of cellulardivision, either through mitosis or meiosis, whereby increased cellnumbers result. In particular embodiments, therapeutic treatments reducecellular growth. Cellular growth refers both to an increase in cell massor size, as well as cellular physiological processes necessary tosupport a cell's life.

Particular conditions that can be treated include various cancers,thyroid diseases (e.g., hyperthyroidism or thyrotoxicosis), blooddisorders (e.g., Polycythemia vera, an excess of red blood cellsproduced in the bone marrow), and cellular proliferation in bloodvessels following balloon angioplasty and/or stent placement (known asrestenosis).

The effectiveness of a therapeutic treatment can be confirmed based on abeneficial change related to the condition following the treatment.

In the context of cancers, therapeutic treatments can decrease thenumber of cancer cells, decrease the number of metastases, decreasetumor volume, increase life expectancy, induce chemo- orradiosensitivity in cancer cells, inhibit angiogenesis near cancercells, inhibit cancer cell proliferation, inhibit tumor growth, preventor reduce metastases, prolong a subject's life, reduce cancer-associatedpain, and/or reduce relapse or re-occurrence of cancer followingtreatment. In particular embodiments, therapeutic treatments reduce,delay, or prevent further metastasis from occurring.

For hyperthyroidism or thyrotoxicosis, therapeutic treatments can aid inthe return of thyroid secreted hormones, such as T3 and T4, to morenormal levels. These hormones can be measured from patient bloodsamples. In particular embodiments, a therapeutic treatment returnsserum levels of T3 and/or T4 to within a normal range (80-180 ng/dl and4.6-12 μg/dl, respectively).

For Polycythemia vera, therapeutic treatments can aid in the return ofred blood cell counts to more normal levels. In particular embodiments,a therapeutic treatment returns the red blood cell count to within anormal range (4.7 to 6.1 million cells/μl).

For restenosis, therapeutic treatments can include the placement ofradionuclides in the region of a vessel where a stent was placed orballoon angioplasty was performed, in order to inhibit the narrowing ofthe vessel due to proliferation of blood vessel cells. Treatment forrestenosis can be deemed effective if normal blood flow through theaffected blood vessel is restored. One test that can be used to diagnoseimproper blood flow is a stress test, which involves physical exercisewhile blood pressure and heart rate are measured. A normal stress testresult means that the patient was able to exercise for a normal lengthof time and at a normal intensity level for their age and gender.Another test that can be performed to diagnose improper blood flow is aCT or MRI angiogram, which involves placement of a dye into thebloodstream and imaging of blood vessels. If restenosis treatment iseffective, the CT or MRI angiogram will reveal normal blood flow throughthe affected vessel.

As indicated previously, particular uses of the chelating platformsdisclosed herein include in imaging and treatment in the same subject.

The actual dose amount administered to a particular subject can bedetermined by a physician, veterinarian, or researcher taking intoaccount parameters such as physical and physiological factors includingbody weight; severity of condition; previous or concurrent therapeuticinterventions; idiopathy of the subject; and route of administration.

In particular embodiments, the total dose of absorbed radiation mayinclude 10-3 grays (Gy), 10-2 Gy, 10-1 Gy, 1 Gy, 5 Gy, 10 Gy, 25 Gy, 50Gy, 75 Gy, 100 Gy, 200 Gy, 300 Gy, 400 Gy, 500 Gy, 600 Gy, 700 Gy, 800Gy, 900 Gy, or 1000 Gy.

Doses of absorbed radiation can be achieved by delivering an appropriateamount of a composition. Exemplary amounts of compositions can include0.05 mg/kg to 5.0 mg/kg administered to a subject per day in one or moredoses. For certain indications, the total daily dose can be 0.05 mg/kgto 3.0 mg/kg administered intravenously to a subject one to three timesa day, including administration of total daily doses of 0.05-3.0,0.1-3.0, 0.5-3.0, 1.0-3.0, 1.5-3.0, 2.0-3.0, 2.5-3.0, and 0.5-3.0mg/kg/day of composition using 60-minute QD, BID, or TID intravenousinfusion dosing. Additional useful doses can often range from 0.1 to 5μg/kg or from 0.5 to 1 μg/kg. In other examples, a dose can include 1μg/kg, 20 μg/kg, 40 μg/kg, 60 μg/kg, 80 μg/kg, 100 μg/kg, 200 μg/kg, 350μg/kg, 500 μg/kg, 700 μg/kg, 0.1 to 5 mg/kg, or from 0.5 to 1 mg/kg. Inother examples, a dose can include 1 mg/kg, 10 mg/kg, 20 mg/kg, 40mg/kg, 60 mg/kg, 80 mg/kg, 100 mg/kg, 200 mg/kg, 400 mg/kg, 500 mg/kg,700 mg/kg, 750 mg/kg, 1000 mg/kg, or more.

Therapeutically effective amounts can be achieved by administeringsingle or multiple doses during the course of an imaging or treatmentregimen (e.g., daily, every other day, every 3 days, every 4 days, every5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly,every 2 months, every 3 months, every 4 months, every 5 months, every 6months, every 7 months, every 8 months, every 9 months, every 10 months,every 11 months, or yearly).

Provided herein are methods for the separation or purification of metalions. Currently available methods for the separation and purification ofmetal ions are either hydrometallurgical processes (liquid-liquidextraction, precipitation, electrodeposition, etc.) or analyticaltechniques (high performance liquid chromatography, ion chromatography,impregnated resins, capillary electrophoresis, mass spectrometry, etc.).The hydrometallurgical processes usually allow one to process importantquantities of materials, are operated at ambient or low pressure butrequire harsh chemical conditions and multiple steps in order to reachthe desire purity. Their recovery yield also rarely reaches 100%. Incontrast, the analytical techniques convey very little quantities, areoperated at very high pressure (such as in HPLC methods) or very lowpressure and high temperature (as in mass spectrometry techniques) andthey are difficult, if not impossible, to scale-up for industrialapplications. Both hydrometallurgical and analytical methods also oftenrequire the use of organic solvents either hydrosoluble or hydrophobic.The new strategy provided herein can be fast, efficient, scalable andoperable at room temperature, ambient pressure and in fully aqueoussolvent.

Experimental results showed that the siderocalin protein is able to bindvarious charged metal complexes through electrostatic interactions.While siderocalin binds tightly to negatively charged complexes, itdoesn't bind to neutral complexes due to a lack of electrostaticinteractions. The general strategy provided is to use siderocalin (orvariants thereof or other proteins) as a platform for the separation ofmetal ions owing to the formation of high-molecular species(Protein-composition (e.g., ligand) metal adduct) versus low-molecularweight species (composition (e.g., ligand)-metal complex that is notrecognized by the protein).

The low-molecular weight composition (e.g., ligand) can be tuned tomatch the charge of the metal ions to separate. For instance, thecomplex of zirconium ion (Zr⁴⁺) with [3,4,3-LI(1,2-HOPO)]⁴⁻ is neutral([Zr(IV)L]⁰) and therefore it is not recognized by siderocalin. Incontrast, the complex of europium ion (Eu³⁺) with [3,4,3-LI(1,2-HOPO)]⁴⁻possesses one negative charge ([Eu(III)L]⁻) and it is recognized andtightly bound by siderocalin. In this example, Zr⁴⁺ and Eu³⁺ ions cantherefore be separated.

In some embodiments, the separation of ions is based on the formation ofhigh-molecular weight versus low-molecular weight species. Theprotein-composition (e.g., ligand) system therefore acts as a sortingdevice at a molecular level. The separation in itself between thelow-molecular weight species and the macromolecular entities can bebased on various fundamental leverages like differences in size, mass,polarity, solubility, etc. Hence, the separation of themetal-composition (e.g., ligand)-protein adducts from the low-molecularweight species can potentially be performed by various techniques suchas (among others) size exclusion chromatography, cut-off filterfiltration, solid-liquid extraction using solid supports grafted withthe protein or the protein-composition (e.g., ligand) adduct, tangentialfiltration, ultra-, micro- or nano-filtration and liquid-liquidextraction. Experimental examples and details for the separation ofdifferent metal ions are provided herein.

Some embodiments provided herein can be used for the separation ofactinides. In some embodiments, the methods provided herein can be usedfor sequestering and separation of radioactive elements. In someembodiments, the protein siderocalin displays a range of affinities withdifferent actinide and lanthanide complexes of natural and syntheticcompositions (e.g., ligands) that can allow new separation approaches.

As noted above, siderocalin (Scn) is an iron-transport protein that alsobinds actinide elements (such as thorium, plutonium, americium, andcurium). In some embodiments, this discovery can be applied as amolecular mechanism through which radioactive elements can be probed andremediated from a contaminated environment.

In some embodiments, provided herein are methods and compositions forthe separation of metal ions of differing charges from one another. Insome embodiments, M³⁺ ions can be separated from M⁴⁺ ions, using asystem comprising the composition (e.g., ligand), such as3,4,3-LI(1,2-HOPO) and the protein siderocalin (or other protein thatselectively binds to a charged (or uncharged) complex). In someembodiments, provided herein are separation processes for M⁴⁺ ions,based on the use of 3,4,3-LI(1,2-HOPO). That is, the resulting complexof metal+composition (e.g., ligand) is either bound, or not, by a largermolecular weight protein. The fact that the metal composition (e.g.,ligand) complex is either bound or not to the larger molecular weightprotein allows one to separate the metal via separating the largerprotein. In some embodiments, the binding of the composition (e.g.,ligand) to the metal allows one to create either a charged complex or aneutral complex (depending upon the combined charge of the ligand andmetal), which in turn is selectively bound (or not) by the protein(e.g., siderocalin). In some embodiments, methods and compositions areprovided for separation between Bk⁴⁺ and other M³⁺ metals.

The applications of the invention are numerous, going from waterpurification to nuclear waste treatment and also portable analysis,production of radiation sources, etc. The process can selectively remove(or enrich) one charged species over a different charge species. Typicalexamples of metal ions separation include, among others, the separationof plutonium (Pu⁴⁺) from adjacent actinide elements (Am³⁺, Cm³⁺, Cf³⁺),the separation of thorium (Th⁴⁺) from actinium (Ac³⁺) or scandium(Sc³⁺), the separation of zirconium (Zr⁴⁺) from yttrium (Y³⁺), theseparation of tin (Sn⁴⁺) from indium (In³⁺), the separation of cerium(Ce⁴⁺) from other lanthanides (Ln³⁺) and the separation of berkelium(Bk⁴⁺) form other actinides (Cf³⁺, Cm³⁺, Am³⁺). The methods andcompositions provided herein can be used in a variety of settings,including, without limitation, for: 1) the mining industry, 2) thenuclear industry, 3) pharmaceutical isotope production industry, and/or4) the chemical industry developing separation supports.

In some embodiments, one can scale the process such that it allows forversatile separation of different metal ions under soft conditions(aqueous environments, ambient pressure and temperature, high yields,separation material recovery for numerous separation cycles, etc.).Based on the experimental results presented herein, one can employ suchprocesses through standard engineering techniques.

As noted above, experimental results have shown that a protein (B. E.Allred, P. B. Rupert, S. S. Gauny, D. D. An, C. Y. Ralston, M.Sturzbecher-Hoehne, R. K. Strong, and R. J. Abergel,“Siderocalin-mediated recognition, sensitization, and cellular uptake ofactinides,” Proc. Natl. Acad. Sci., vol. 112, no. 33, pp. 10342-10347,August 2015), called siderocalin, is able to bind various charged metalcomplexes. The metal complexes have the general formula [ML]^(n−) andsiderocalin is able to bind these complexes due to electrostaticinteractions. The final adduct “metal-ligand-protein” is referredhereafter as “Scn[ML]” (Scn standing for siderocalin, M for metal and Lfor ligand, such as one of the compositions provided herein). Theprotein recognizes the metal complexes of the synthetic ligands[3,4,3-LI(1,2-HOPO)]⁴⁻ or [3,4,3-LI-CAM]⁸⁻ (shown below).

Formulas for 3,4,3-LI(1,2-HOPO) (top) and 3,4,3-LI-CAM (bottom).

While siderocalin binds tightly to the negatively charged complexes, itdoesn't bind to the neutral complexes due to a lack of electrostaticinteractions. In some embodiments provided herein, the general strategyis to use siderocalin (or other proteins) as a platform for theseparation of metal ions owing to the formation of high-molecularspecies versus low-molecular weight species. The discrimination betweentwo or more ions can be due to fundamentally different processes, someembodiments of which are summarized in FIG. 17.

FIG. 17 is a flow chart of some embodiments of the processes leading tothe formation of a low-molecular weight species or high-molecular weightspecies. This separated arrangement provides the ability to separatevarious species. A metal ion of interest (to either enrich or remove) iscombined with a composition (e.g., ligand) (having the opposite charge).Where there is an interaction between the two, to form a complex, thecomplex can either be uncharged (resulting in no recognition or adequatebinding by the protein (such as siderocalin), or charged, which in turncan be recognized by the protein (bound by it) resulting in the metalbeing associated with the higher molecular weight species or notrecognized by the protein (resulting in the metal being in thelow-molecular weight species. Thus, in some embodiments, selection ofthe metal can occur or go through one or more of the steps outlined inFIG. 17. In some embodiments, selection is based upon formation of acharged complex and then selection of the higher molecular weightspecies over the lower molecular weight species (to obtain the metal).In some embodiments, when the metal to be selected results in a neutralcomplex, then the selection is such that the composition (e.g., ligand)binds to the metal, resulting in a neutral complex, which is thenobtained by selecting the low-molecular weight species from the sample.In some embodiments, selection occurs by a charged complex beinggenerated and the protein recognizing the complex (and one collects thehigh-molecular weight species).

In some embodiments, the low-molecular weight composition (e.g., ligand)can be tuned to match the charge of the metal ions to separate. Forinstance, the complex of zirconium ion (Zr⁴⁺) with[3,4,3-LI(1,2-HOPO)]⁴⁻ is neutral ([Zr(IV)L]⁰) and therefore it is notrecognized by siderocalin. In contrast, the complex of europium ion(Eu³⁺) with [3,4,3-LI(1,2-HOPO)]⁴⁻ possesses one negative charge([Eu(III)L]⁻) and it is recognized and tightly bound by siderocalin.Thus, Zr⁴⁺ and Eu³⁺ ions (or other ions with similar charge differences)can therefore be separated.

The applications of the invention are numerous, going from waterpurification to nuclear waste treatment and also portable analysis,production of radiation sources, etc. Examples of metal ions separationwould include (but are not limited to), the separation of plutonium(Pu⁴⁺) from adjacent actinide elements (Am³⁺, Cm³⁺, Cf³⁺), theseparation of thorium (Th⁴⁺) from actinium (Ac³⁺) or scandium (Sc³⁺),the separation of zirconium (Zr⁴⁺) from yttrium (Y³⁺), the separation oftin (Sn⁴⁺) from indium (In³⁺), the separation of cerium (Ce⁴⁺) fromother lanthanides (Ln³⁺) and the separation of berkelium (Bk⁴⁺) formother actinides (Cf³⁺, Cm³⁺, Am³⁺). In some embodiments, any first metalcan be separated from a second metal as long as there is a difference incharge between the first metal and the second metal. In someembodiments, the first metal can be separated from two, three, four,five or more other metals, as long as the first metal differs in chargefrom the other metals.

In some embodiments, the methods provided herein can be performed atroom temperature, and/or ambient temperature, and/or in a one-stepprocess, and/or under mild chemical conditions (e.g., fully aqueoussolvent, pH 7.4). In some embodiments, there are no volatile elements inthe processing. In some embodiments, there are volatile elements in theprocessing.

In some embodiments, separation can be achieved between any two metalsthat differ in charge, including those above, below in the examples,and/or any of the following pairs Ac³⁺/Th⁴⁺, Bk⁴⁺/Cf³⁺, M⁴⁺/M³⁺, andM⁴⁺/M²⁺.

In some embodiments, the enrichment/purification method can comprisecontacting a liquid comprising a plurality of metal ions with acomposition as described herein, under conditions sufficient to form ametal ion-composition complex comprising a metal ion of the plurality ofmetal ions. The method can further comprise separating a first fractionof the mixture enriched for the metal ion-composition complex from asecond fraction depleted for the metal ion-composition complex, whereinthe first fraction is enriched for a first metal ion that has a chargethat is different from a charge of a second metal ion enriched in thesecond fraction.

In some embodiments, the method isolates Bk⁴⁺ from a mixture. The methodcan comprise contacting a first mixture comprising Bk⁴⁺ and a trivalentmetal ion with a composition as described herein under conditionssufficient to form a complex comprising the trivalent metal ion and thecomposition. The method can further include separating the complex fromthe first mixture to generate a second mixture depleted for thetrivalent metal ion and chromatographically isolating the Bk⁴⁺ in thesecond mixture.

In some embodiments, the method reclaims an actinide from a sample. Themethod comprises obtaining an aqueous sample comprising, or suspected ofcomprising, an actinide, contacting the sample with a composition asdescribed herein to generate a mixture under conditions sufficient toform a complex comprising the actinide and the composition; andseparating the complex from the mixture.

In some embodiments, separating is based on molecular weight. In someembodiments, the separating comprises size-exclusion chromatography oraffinity chromatography.

In some embodiments, a first fraction is enriched for a trivalent metalion or a divalent metal ion, and a second fraction is enriched for atetravalent metal ion. In some embodiments, a first fraction is enrichedfor a metal ion selected from the group consisting of: actinides,lanthanides, Ac³⁺, Sc³⁺, Y³⁺, and In³⁺. In some embodiments, a secondfraction is enriched for a metal ion selected from the group consistingof: Pu⁴⁺, Np⁴⁺, Th⁴⁺, Zr⁴⁺, Sn⁴⁺, Ce⁴⁺, and Bk⁴⁺. In some embodiments, afirst fraction is enriched for a metal ion selected from the groupconsisting of Am³⁺, Cm³⁺, Bk³⁺ and Cf³⁺, and a second fraction comprisesPu⁴⁺. In some embodiments, a first fraction is enriched for Ac³⁺ and/orSc³⁺, and a second fraction is enriched for Th⁴⁺. In some embodiments, afirst fraction is enriched for Eu³⁺ or Y³⁺, and a second fraction isenriched for Zr⁴⁺. In some embodiments, a first fraction is enriched forIn³⁺, and a second fraction is enriched for Sn⁴⁺. In some embodiments, afirst fraction is enriched for a lanthanide, and a second fraction isenriched for Ce⁴⁺. In some embodiments, a first fraction is enriched forTm³⁺. In some embodiments, a first fraction is enriched for an actinide,and a second fraction is enriched for Bk⁴⁺. In some embodiments, a firstfraction is enriched for a metal ion selected from the group consistingof Am³⁺, Cm³⁺, and Cf³⁺.

In some embodiments, a first mixture further comprises one or moreactinides selected from the group consisting of: Cm³⁺, Am³⁺, Cf³⁺, Th⁴⁺,Np⁴⁺, Pu⁴⁺, and Ce⁴⁺. In some embodiments, a mixture is prepared byneutron irradiation of Pu, Am or Cm. In some embodiments, thecomposition comprises a hydroxypyridonate ligand. In some embodiments,the composition comprises 3,4,3-LI(1,2-HOPO).

In some embodiments, the actinide comprises Am³⁺ and/or Cm³⁺.

In some embodiments, the sample is derived from a river, ocean, lake,soil, or industrial run off. In some embodiments, the sample is anindustrial sample. In some embodiments, the composition comprises ahydroxypyridonate ligand. In some embodiments, the composition comprises3,4,3-LI(1,2-HOPO).

Current processes to separate Bk from Am, Cm, Cf, and fission productsafter its production by neutron irradiation of Pu, Am, or Cm targets,necessitate numerous steps and use strong oxidizers such as sodiumbromate to segregate Bk(IV) from the non-tetravalent ions. Thenon-recognition of [Bk^(IV)3,4,3-LI(1,2-HOPO)] by Scn suggestsinnovative procedures to separate Bk from M(III) ions could involvepassing a 3,4,3-LI(1,2-HOPO) solution of the irradiated mixture througha Scn-containing medium, followed by size-exclusion discrimination.However, the separation of Bk(IV) from other M(IV) ions potentiallypresent during Bk production, namely Ce, Th and Pu, also present achallenge. In current production-purification processes, the Ce—Bk pairis difficult due to the almost-identical redox properties of the twoelements, which has led to complicated solvent extraction or ionexchange techniques. FIG. 26 displays the relative retention of variousM(IV) complexes of 3,4,3-LI(1,2-HOPO) on a classical C18 LC column. Theretention time of the Bk complex falls between those of Zr(IV) andCe(IV), trending with the ionic radii of the metals, whenocta-coordinated (0.84, 0.93, and 0.97 pm for Zr⁴⁺, Bk⁴⁺ and Ce⁴⁺respectively). Without actual optimization, [Bk^(IV)3,4,3-LI(1,2-HOPO)]was easily discriminated from its Ce, Th and Pu analogs. Hence, atwo-step separation process is sufficient for separating Bk from allother Ln and An ions, with step 1 sequestering 3⁺ ions based on Scnselectivity towards [M^(III)3,4,3-LI(1,2-HOPO)]⁻ complexes, and step 2separating Bk from 4⁺ ions under classical chromatography. This entireprocedure is mono-phasic, operated at room temperature and does notrequire any liquid-liquid extraction step or introduce additionalnon-volatile elements.

In some embodiments, further separation of metals with the same valenceor oxidation state (such as M4+ from M4+, M3+ from M3+ or M2+ from M2+)can be achieved through classical liquid chromatography, as thecomplexes formed between the metal and ligands described in theinvention (including 3,4,3-LI(1,2-HOPO)) exhibit different retentiontimes on standard chromatographic columns (see FIG. 26).

EXEMPLARY EMBODIMENTS

-   -   1. A method of treating a subject in need thereof comprising        administering to the subject a therapeutically effective amount        of a composition including a structure

-   -   wherein:    -   (i) A1, A2, A3, and A4, individually, include a CAM group, a        1,2-HOPO group, or a HA group;    -   (ii) B1, B2, B3, and B4, individually, include an amide group or        an amine group;    -   (iii) at least one of C1, C2, C3, C4, C5, or C6, individually,        include SH, C(═O)OH, or NH₂;    -   (iv) at least another one of C1, C2, C3, C4, C5, or C6 is        optional;    -   (v) at least one of L1, L2, L3, L4, L5, L6, L7, L8, L9, L10,        L11, L12, or L13, individually, include H, an alkyl group having        no greater than 10 carbon atoms, an alkylamino group having no        greater than 10 carbon atoms and no greater than 2 nitrogen        atoms; an alkyl ether group having no greater than 10 carbon        atoms, a hydroxy ester group, or an alkyl ester group having no        greater than 10 carbon atoms; and    -   (vi) at least one of L1, L5, L6, L7, L8, L9, L10, L11, L12, or        L13 is optional;    -   wherein the structure of the composition is bound to a        siderocalin and a metal, thereby treating the subject.    -   2. A method of embodiment 1, wherein at least another one of L2,        L3, or L4, individually, include an amine group or an amide        group.    -   3. A method of embodiment 1 or embodiment 2, wherein L1, C1, L7,        C2, L9, C3, L11, C4, and L13, C5 are absent, L5 includes an        unsubstituted alkyl group having no greater than 5 carbon atoms,        and C6 includes SH, C(═O)OH, or NH₂.    -   4. A method of embodiment 3, wherein L2, L3, L4, L6, L8, L10,        and L12, individually, include an unsubstituted alkyl group        having no greater than 5 carbon atoms.    -   5. A method of embodiment 4, wherein A1 includes a CAM group or        a HOPO group; A2 includes a HA group, A3 includes a HA group,        and A4 includes a CAM group, a HOPO group, or a HA group.    -   6. A method of any one of embodiments 1-5, wherein at least one        of L2, L3, or L4, individually, include an alkylamino group.    -   7. A method of embodiment 1, wherein B1, B2, and B3,        individually, include an amide group and B4 includes an amino        group, L2 and L3 include an amino group, and L4 includes an alky        group having no greater than 5 carbon atoms    -   8. A method of embodiment 7, wherein:        C1, C2, C3, C4, C5, L1, A1, A2, A3, L1, L6, L7, L8, L9, L10,        L11, L12, and L13 are absent,        A4 includes a CAM group, a HOPO group, or a HA group; and        L5 includes an alkyl group having no greater than 5 carbon        atoms.    -   9. A method of embodiment 1, wherein 1, B2, and B3,        individually, include an amide group and B4 includes an amide        group, L2 and L3, individually, include an amino group, and L4        includes an alky group having no greater than 5 carbon atoms.    -   10. A method of embodiment 9, wherein C1, C2, C3, C4, C5, A1,        A2, A3, L1, L6, L7, L8, L9, L10, L11, and L13 are absent, L12        includes an amino group, L5 includes an ether group having no        greater than 10 carbon atoms, and A4 includes a CAM group, a        HOPO group, or a HA group.    -   11. A method of embodiment 1, wherein C1, C2, C5, C6, L1, L2,        L3, L4, L5, L7, L13, B2, and B4 are absent, 1 and B3,        individually, include an amide group, L6, L8, L10, and L12,        individually, include an amino group, A1, A2, A3, and A4,        individually, include a CAM group, a HOPO group, or a HA group,        L9 and 11, individually, include an alkyl group having no        greater than 5 carbon atoms.    -   12. A method of treating a subject in need thereof comprising        administering to the subject a therapeutically effective amount        of a composition including a structure:

-   -   -   wherein:        -   at least one of R₁, R₂, R₃, R₄, and R₅, individually,            include a CAM group, a HA group, or a 1,2-HOPO group;        -   at least another one of R₁, R₂, R₃, R₄, and R₅,            individually, include H or an alkyl group having from 1 to            10 carbon atoms;        -   R₆ includes (i) H, (ii) an alkyl group having from 1 to 10            carbon atoms, or (iii) an alkyl group having from 1 to 10            carbon atoms and substituted by at least one of SH, NH₂, or            C(═O)OH;        -   m can be from 1 to 6;        -   n can be from 1 to 6;        -   can be from 1 to 6;            wherein the structure of the composition is bound to a            siderocalin and a metal, thereby treating the subject.

    -   13. A method of embodiment 12, including a structure:

wherein:at least one of R₁, R₃, R₄, or R₅ R₁, R₂, R₃, R₄, and R₅, individually,include a CAM group, a HA group, or a 1,2-HOPO group;optionally, another one of R₁, R₃, R₄, or R₅ R₁, R₂, R₃, R₄, and R₅,individually, include H or an alkyl group having from 1 to 10 carbonatoms;R₂ includes H or an alkyl group including from 1 to 5 carbon atoms;R₇ includes SH, C(═O)OH, or NH₂; andp is from 0 to 4.

-   -   14. A method of embodiment 13, wherein:        R₁ includes a CAM group or a 1,2-HOPO group;        R₃ and R₄, individually, include a HA group; and        R₅ includes a CAM group, a 1,2-HOPO group, or a HA group.    -   15. A method of embodiment 12, including a structure:

wherein:R₇ includes SH, NH₂, or C(═O)OH;R₂, R₈, and R₉, individually, include H, OH, or an alkyl group includingfrom 1 to 5 carbon atoms; andp is from 0 to 4.

-   -   16. A method of embodiment 12, including a structure:

wherein:R₇ includes SH, NH₂, or C(═O)OH;R₂, R₈, and R₉, individually, include H, OH, or an alkyl group includingfrom 1 to 5 carbon atoms; andp is from 0 to 4.

-   -   17. A method of embodiment 12, including a structure:

wherein:R₇ includes SH, NH₂, or C(═O)OH;R₂, R₈, R₉, and R₁₀, individually, include H, OH, or an alkyl groupincluding from 1 to 5 carbon atoms; andp is from 0 to 4.

-   -   18. A method of embodiment 12, including a structure:

wherein:R₇ includes SH, NH₂, or C(═O)OH;R₂, R₈, and R₉, individually, include H, OH, or an alkyl group includingfrom 1 to 5 carbon atoms; andp is from 0 to 4.

-   -   19. A method of embodiment 12, including a structure:

wherein:R₇ includes SH, NH₂, or C(═O)OH;R₂, R₈, and R₉, individually, include H, OH, or an alkyl group includingfrom 1 to 5 carbon atoms; andp is from 0 to 4.

-   -   20. A method of embodiment 12, including a structure:

wherein:R₇ includes SH, NH₂, or C(═O)OH;R₂, R₈, R₉, and R₁₀, individually, include H, OH, or an alkyl groupincluding from 1 to 5 carbon atoms; andp is from 0 to 4.

-   -   21. A method of embodiment 12, including a structure:

-   -   22. A method of embodiment 12, including a structure:

-   -   23. A method of treating a subject in need thereof comprising        administering to the subject a therapeutically effective amount        of a composition including a structure:

-   -   -   wherein:        -   at least one of R₁₁, R₁₂, R₁₃, or R₁₅, individually, include            a CAM group, a HA group, or a 1,2-HOPO group;        -   optionally, at least another one of R₁₁, R₁₂, R₁₃, or R₁₅,            individually, include H, OH, or an alkyl group having from 1            to 10 carbon atoms;        -   R₁₇ includes SH, NH₂, or C(═O)OH;        -   R₂, R₁₄, and R₁₆, individually, include H, OH, or an alkyl            group having from 1 to 10 carbon atoms; and        -   r can be from 0 to 6            wherein the structure of the composition is bound to a            siderocalin and a metal, thereby treating the subject.

    -   24. A method of embodiment 23, wherein:        R₁₁ includes a CAM group or a 1,2-HOPO group;        R₁₂ and R₁₅, individually, include a HA group; and        R₁₃ includes a CAM group, a 1,2-HOPO group, or a HA group.

    -   25. A method of embodiment 23, including a structure:

wherein:R₂, R₁₄, R₁₆, R₁₈, and R₁₉, individually, include H, OH, or an alkylgroup having from 1 to 10 carbon atoms;R₁₇ includes SH, NH₂, or C(═O)OH; andr can be from 0 to 4.

-   -   26. A method of embodiment 23, including a structure:

whereinR₂, R₁₄, R₁₆, R₁₈, and R₁₉, individually, include H, OH, or an alkylgroup having from 1 to 10 carbon atoms;R₁₇ includes SH, NH₂, or C(═O)OH; andr is from 0 to 4.

-   -   27. A method of embodiment 23, including a structure:

wherein:R₂, R₁₄, R₁₆, R₁₈, R₁₉, and R₂₀, individually, include H, OH, or analkyl group having from 1 to 10 carbon atoms;R₁₇ includes SH, NH₂, or C(═O)OH; andr can be from 0 to 4.

-   -   28. A method of embodiment 23, including a structure:

wherein:R₂, R₁₄, R₁₆, R₁₈, R₁₉, and R₂₀, individually, include H, OH, or analkyl group having from 1 to 10 carbon atoms;R₁₇ includes SH, NH₂, or C(═O)OH; andr can be from 0 to 4.

-   -   29. A method of embodiment 23, including a structure:

wherein:R₂, R₁₄, R₁₆, R₁₈, and R₁₉, individually, include H, OH, or an alkylgroup having from 1 to 10 carbon atoms;R₁₇ includes SH, NH₂, or C(═O)OH; andr is from 0 to 4.

-   -   30. A method of embodiment 23, including a structure:

wherein:R₂, R₁₄, R₁₆, R₁₈, and R₁₉, individually, include H, OH, or an alkylgroup having from 1 to 10 carbon atoms;R₁₇ includes SH, NH₂, or C(═O)OH;r is from 0 to 4.

-   -   31. A method of treating a subject in need thereof comprising        administering to the subject a therapeutically effective amount        of a composition including a structure:

-   -   -   wherein:        -   R₂₁ and R₂₂, individually, include H, OH, or an alkyl group            having from 1 to 10 carbon atoms;        -   R₂₃ includes H, OH, an alkyl group having from 1 to 10            carbon atoms, or (CH₂)_(e)R_(a),        -   where R_(a) is SH, C(═O)OH, or NH₂;        -   R₂₄ includes a substituent having a CAM group, a 1,2-HOPO            group, or a HA group;        -   a, b, and c, individually, are from 1 to 10;        -   d is from 1 to 4; and        -   e is from 1 to 10;            wherein the structure of the composition is bound to a            siderocalin and a metal, thereby treating the subject.

    -   32. A method of embodiment 31, wherein R₂₄ includes a        substituent having SH, C(═O)OH, or NH₂.

    -   33. A method of embodiment 31, including a structure:

wherein:R₂₅, R₂₆, and R₂₇, individually, include H, OH, or an alkyl group havingfrom 1 to 10 carbon atoms;R₂₈ includes H, an alkyl group having from 1 to 5 carbon atoms, SH, NH₂,or C(═O)OH; ands is from 0 to 4.

-   -   34. A method of embodiment 31, including a structure:

wherein:R₂₅, R₂₆, R₂₇, and R₃₀, individually, include H, OH, or an alkyl grouphaving from 1 to 10 carbon atoms;R₂₈ and R₂₉, individually, include H, an alkyl group having from 1 to 5carbon atoms, SH, NH₂, or C(═O)OH;s is from 0 to 4; andt is from 0 to 4.

-   -   35. A method of embodiment 31, including a structure:

wherein:R₂₅, R₂₆, R₂₇, and R₃₀, individually, include H, OH, or an alkyl grouphaving from 1 to 10 carbon atoms;R₂₈ and R₂₉, individually, include H, an alkyl group having from 1 to 5carbon atoms, SH, NH₂, or C(═O)OH;s is from 0 to 4; andt is from 0 to 4.

-   -   36. A method of embodiment 31, including a structure:

wherein:R₂₅, R₂₆, R₂₇, R₃₀, and R₃₁, individually, include H, OH, or an alkylgroup having from 1 to 10 carbon atoms;R₂₈ and R₂₉, individually, include H, an alkyl group having from 1 to 5carbon atoms, SH, NH₂, or C(═O)OH;s is from 0 to 4; andt is from 0 to 4.

-   -   37. A method of embodiment 31, including a structure:

whereinR₂₅, R₂₆, and R₂₇, individually, include H, OH, or an alkyl group havingfrom 1 to 10 carbon atoms;R₂₈ includes H, an alkyl group having from 1 to 5 carbon atoms, SH, NH₂,or C(═O)OH; ands is from 0 to 4.

-   -   38. A method of embodiment 31, including a structure:

wherein:R₂₅, R₂₆, R₂₇, and R₃₂, individually, include H, OH, or an alkyl grouphaving from 1 to 10 carbon atoms;R₂₈ includes H, an alkyl group having from 1 to 5 carbon atoms, SH, NH₂,or C(═O)OH; ands is from 0 to 4.

-   -   39. A method of treating a subject in need thereof comprising        administering to the subject a therapeutically effective amount        of a composition including a structure:

-   -   -   wherein:        -   A, B, C, and D, individually, include one or more amide            groups, one or more amine groups, or an alkyl group having            from 1 to 10 carbon atoms;        -   R₃₃, R₃₄, R₃₅, and R₃₆, individually, include a CAM group, a            1,2-HOPO group, or a HA group; and        -   g, h, i, and j, individually, are from 1 to 10;            wherein the structure of the composition is bound to a            siderocalin and a metal, thereby treating the subject.

    -   40. A method of embodiment 39, including a structure:

wherein:R₃₇ and R₄₂, individually, include H, an alkyl group having from 1 to 5carbon atoms, SH, C(═O)OH, or NH₂;R₃₈, R₃₉, R₄₀, and R₄₁, individually, include H, OH, or an alkyl grouphaving from 1 to 5 carbon atoms; andu and v, individually, are from 0 to 5.

-   -   41. A method of embodiment 39, including a structure:

wherein:R₃₇ and R₄₂, individually, include H, an alkyl group having from 1 to 5carbon atoms, SH, C(═O)OH, or NH₂;R₃₈, R₃₉, R₄₀, and R₄₁, individually, include H, OH, or an alkyl grouphaving from 1 to 5 carbon atoms; andu and v, individually, are from 0 to 5.

-   -   42. A method of embodiment 39, including a structure:

wherein:R₃₇ and R₄₂, individually, include H, an alkyl group having from 1 to 5carbon atoms, SH, C(═O)OH, or NH₂;R₃₈, R₃₉, R₄₀, and R₄₁, individually, include H, OH, or an alkyl grouphaving from 1 to 5 carbon atoms; andu and v, individually, are from 0 to 5.

-   -   43. A method of any one of embodiments 1-42, wherein the        siderocalin includes any of SEQ ID NOs: 1-25.    -   44. A method of any one of embodiments 1-43, wherein the        composition is bound to:        position K125 of siderocalin;        position K134 of siderocalin, or        both position K125 and position K134 of siderocalin.    -   45. A method of any one of embodiments 1-44, wherein the metal        is a radionuclide.    -   46. A method of embodiment 45, wherein the radionuclide includes        ²²⁵Ac, ²²⁶Ac, ²²⁸Ac, ¹⁰⁵Ag, ¹⁰⁶mAg, ¹¹⁰mAg, ¹¹¹Ag, ¹¹²Ag, ¹¹³Ag,        ²³⁹Am, 240 Am, ²⁴²Am, ²⁴⁴Am, ³⁷Ar, ⁷¹As, ⁷²As, ⁷³As, ⁷⁴As, ⁷⁶As,        ⁷⁷As, ²⁰⁹At, ²¹⁰At, ¹⁹¹Au, ¹⁹²Au, ¹⁹³Au, ¹⁹⁴Au, ¹⁹⁵Au, ¹⁹⁶Au,        ¹⁹⁶m²Au, ¹⁹⁸Au, ¹⁹⁸mAu, ¹⁹⁹Au, ²⁰⁰mAu, ¹²⁸Ba, ¹³¹Ba, ¹³³mBa,        ¹³⁵mBa, ¹⁴⁰Ba, ⁷Be, ²⁰³Bi, ²⁰⁴Bi, ²⁰⁵Bi, ²⁰⁶Bi, ²¹⁰Bi, ²¹²Bi,        ²⁴³Bk, ²⁴⁴Bk, ²⁴⁵Bk, ²⁴⁶Bk, ²⁴⁸mBk, ²⁵⁰Bk, ⁷⁶Br, ⁷⁷Br, ⁸⁰mBr,        ⁸²Br, ¹¹C, ¹⁴C, ⁴⁵Ca, ⁴⁷Ca, ¹⁰⁷Cd, ¹¹⁵Cd, ¹¹⁵mCd, ¹¹⁷mCd, ¹³²Ce,        ¹³³mCe, ¹³⁴Ce, ¹³⁵Ce, ¹³⁷Ce, ¹³⁷mCe, ¹³⁹Ce, ¹⁴¹Ce, ¹⁴³Ce, ¹⁴⁴Ce,        ²⁴⁶Cf, ²⁴⁷Cf, ²⁵³Cf, ²⁵⁴Cf, ²⁴⁰Cm, ²⁴¹Cm, ²⁴²Cm, ²⁵²Cm, ⁵⁵Co,        ⁵⁶Co, ⁵⁷Co, ⁵⁸Co, ⁵⁸mCo, ⁶⁰Co, ⁴⁸Cr, ⁵¹Cr, ¹²⁷Cs, ¹²⁹Cs, ¹³¹Cs,        ¹³²Cs, ¹³⁶Cs, ¹³⁷Cs, ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ¹⁵³Dy, ¹⁵⁵Dy,        ¹⁵⁷Dy, ¹⁵⁹Dy, ¹⁶⁵Dy, ¹⁶⁶Dy, ¹⁶⁰Er, ¹⁶¹Er, ¹⁶⁵Er, ¹⁶⁹Er, ¹⁷¹Er,        ¹⁷²Er, ²⁵⁰Es ²⁵¹Es, ²⁵³Es, ²⁵⁴Es, ²⁵⁴mEs, ²⁵⁵Es, ²⁵⁶mEs, ¹⁴⁵Eu,        ¹⁴⁶Eu, ¹⁴⁷Eu, ¹⁴⁸Eu, ¹⁴⁹Eu, ¹⁵⁰mEu, ¹⁵²mEu, ¹⁵⁶Eu, ¹⁵⁷Eu, ⁵²Fe,        ⁵⁹Fe, ²⁵¹Fm, ²⁵²Fm, ²⁵³Fm, ²⁵⁴Fm, ²⁵⁵Fm, ²⁵⁷Fm, ⁶⁶Ga, ⁶⁷Ga,        ⁶⁸Ga, ⁷²Ga, ⁷³Ga, ¹⁴⁶Gd, ¹⁴⁷Gd, ¹⁴⁹Gd, ¹⁵¹Gd, ¹⁵³Gd, ¹⁵⁹Gd,        ⁶⁸Ge, ⁶⁹Ge, ⁷¹Ge, ⁷⁷Ge, ¹⁷⁰Hf, ¹⁷¹Hf, ¹⁷³Hf, ¹⁷⁵Hf, ¹⁷⁹m²Hf,        ¹⁸⁰mHf, ¹⁸¹Hf, ¹⁸⁴Hf, ¹⁹²Hg, ¹⁹³Hg, ¹⁹³mHg, ¹⁹⁵Hg, ¹⁹⁵mHg,        ¹⁹⁷Hg, ¹⁹⁷mHg, ²⁰³Hg, ¹⁶⁰mHo, ¹⁶⁶Ho, ¹⁶⁷Ho, ¹²³I, ¹²⁴I, ¹²⁶I,        ¹³⁰I, ¹³²I, ¹³³I, ¹³⁵I, ¹⁰⁹In, ¹¹⁰In, ¹¹¹In, ¹¹⁴mIn, ¹¹⁵m In,        ¹⁸⁴Ir, ¹⁸⁵Ir, ¹⁸⁶Ir, ¹⁸⁷Ir, ¹⁸⁸Ir, ¹⁸⁹Ir, ¹⁹⁰Ir, ¹⁹⁰m²Ir, ¹⁹²Ir,        ¹⁹³m Ir, ¹⁹⁴Ir, ¹⁹⁴m²Ir, ¹⁹⁵m Ir, ⁴²K, ⁴³K, ⁷⁶Kr, ⁷⁹Kr, ⁸¹mKr,        ⁸⁵mKr, ¹³²La, ¹³³La, ¹³⁵La, ¹⁴⁰La, ¹⁴¹La, ²⁶²Lr, ¹⁶⁹Lu, ¹⁷⁰Lu,        ¹⁷¹Lu, ¹⁷²Lu, ¹⁷⁴mLu, ¹⁷⁶mLu, ¹⁷⁷Lu, ¹⁷⁷mLu, ¹⁷⁹Lu, ²⁵⁷Md,        ²⁵⁸Md, ²⁶⁰Md, ²⁸Mg, ⁵²Mn, ⁹⁰Mo, ⁹³mMo, ⁹⁹Mo, ¹³N, ²⁴Na, ⁹⁰Nb,        ⁹¹mNb, ⁹²mNb, ⁹⁵Nb, ⁹⁵mNb, ⁹⁶Nb, ¹³⁸Nd, ¹³⁹mNd, ¹⁴⁰Nd, ¹⁴⁷Nd,        ⁵⁶Ni, ⁵⁷Ni, ⁶⁶Ni, ²³⁴Np, ²³⁶mNp, ²³⁸Np, ²³⁹Np, ¹⁵O, ¹⁸²Os,        ¹⁸³Os, ¹⁸³mOs, ¹⁸⁵Os, ¹⁸⁹mOs, ¹⁹¹Os, ¹⁹¹mOs, ¹⁹³Os, ³²P, ³³P,        ²²⁸Pa, ²²⁹Pa, ²³⁰Pa, ²³²Pa, ²³³Pa, ²³⁴Pa, ²⁰⁰Pb, ²⁰¹Pb, ²⁰²mPb,        ²⁰³Pb, ²⁰⁹Pb, ²¹²Pb, ¹⁰⁰Pd, ¹⁰¹Pd, ¹⁰³Pd, ¹⁰⁹Pd, ¹¹¹mPd, ¹¹²Pd,        ¹⁴³Pm, ¹⁴⁸Pm, ¹⁴⁸mPm, ¹⁴⁹Pm, ¹⁵¹Pm, ²⁰⁴Po, ²⁰⁶Po, ²⁰⁷Po, ²¹⁰Po,        ¹³⁹Pr, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁵Pr, ¹⁸⁸Pt, ¹⁸⁹Pt, ¹⁹¹Pt, ¹⁹³mPt, ¹⁹⁵mPt,        ¹⁹⁷Pt, ²⁰⁰Pt, ²⁰²Pt, ²³⁴Pu, ²³⁷Pu, ²⁴³Pu, ²⁴⁵Pu, ²⁴⁶Pu, ²⁴⁷Pu,        ²²³Ra, ²²⁴Ra, ²²⁵Ra, ⁸¹Rb, ⁸²Rb, ⁸²mRb, ⁸³Rb, ⁸⁴Rb, ⁸⁶Rb, ¹⁸¹Re,        ¹⁸²Re, ¹⁸²mRe, ¹⁸³Re, ¹⁸⁴Re, ¹⁸⁴mRe, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re,        ¹⁹⁰mRe, ⁹⁹Rh, ⁹⁹mRh, ¹⁰⁰Rh, ¹⁰¹mRh, ¹⁰²Rh, ¹⁰³mRh, ¹⁰⁵Rh, ²¹¹Rn,        ²²²Rn, ⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru, ³⁵S, ¹¹⁸mSb, ¹¹⁹Sb, ¹²⁰Sb, ¹²⁰mSb,        ¹²²Sb, ¹²⁴Sb, ¹²⁶Sb, ¹²⁷Sb, ¹²⁸Sb, ¹²⁹Sb, ⁴³Sc, ⁴⁴Sc, ⁴⁴mSc,        ⁴⁶Sc, ⁴⁷Sc, ⁴⁸Sc, ⁷²Se, ⁷³Se, ⁷⁵Se, ¹⁵³Sm, ¹⁵⁶Sm, ¹¹⁰Sn, ¹¹³Sn,        ¹¹⁷mSn, ¹¹⁹mSn, ¹²¹Sn, ¹²³Sn, ¹²⁵Sn, ⁸²Sr, ⁸³Sr, ⁸⁵Sr, ⁸⁹Sr,        ⁹¹Sr, ¹⁷³Ta, ¹⁷⁵Ta, ¹⁷⁶Ta, ¹⁷⁷Ta, ¹⁸⁰Ta, ¹⁸²Ta, ¹⁸³Ta, ¹⁸⁴Ta,        ¹⁴⁹Tb, ¹⁵⁰Tb, ¹⁵¹Tb, ¹⁵²Tb, ¹⁵³Tb, ¹⁵⁴Tb, ¹⁵⁴mTb, ¹⁵⁴m²Tb,        ¹⁵⁵Tb, ¹⁵⁶Tb, ¹⁵⁶mTb, ¹⁵⁶m²Tb, ¹⁶⁰Tb, ¹⁶¹Tb, ⁹⁴Tc, ⁹⁵Tc, ⁹⁵mTc,        ⁹⁶Tc, ⁹⁷mTc, ⁹⁹mTc, ¹¹⁸Te, ¹¹⁹Te, ¹¹⁹mTe, ¹²¹Te, ¹²¹ mTe,        ¹²³mTe, ¹²⁵mTe, ¹²⁷Te, ¹²⁷mTe, ¹²⁹mTe, ¹³¹ mTe, ¹³²Te, ²²⁷Th,        ²³¹Th, ²³⁴Th, ⁴⁵Ti, ¹⁹⁸Tl, ¹⁹⁹Tl, ²⁰⁰Tl, ²⁰¹Tl, ²⁰²Tl, ²⁰⁴Tl,        ¹⁶⁵Tm, ¹⁶⁶Tm, ¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁷⁰Tm, ¹⁷²Tm, ¹⁷³Tm, ²³⁰U, ²³¹U,        ²³⁷U, ²⁴⁰U, ⁴⁸V, ¹⁷W, ¹⁸¹W, ¹⁸⁵W, ¹⁸⁷W, ¹⁸⁸W, ¹²²Xe, ¹²⁵Xe,        ¹²⁷Xe, ¹²⁹mXe, ¹³¹mXe, ¹³³Xe, ¹³³mXe, ¹³⁵Xe, ⁸⁵mY, ⁸⁶Y, ⁸⁷Y,        ⁸⁷mY, ⁸⁸Y, ⁹⁰Y, ⁹⁰mY, ⁹¹Y, ⁹²Y, ⁹³Y, ¹⁶⁶Yb, ¹⁶⁹Yb, ¹⁷⁵Yb, ⁶²Zn,        ⁶⁵Zn, ⁶⁹mZn, ⁷¹mZn, ⁷²Zn, ⁸⁶Zr, ⁸⁸Zr, ⁸⁹Zr, ⁹⁵Zr, and/or ⁹⁷Zr.    -   47. A method of embodiment 45, wherein the radionuclide includes        ⁹⁰Y, ⁶⁷Cu, ²¹³Bi, ²¹²Bi, ¹⁸⁶Re, ⁶⁷Cu, ⁹⁰Y, ²¹³Bi, ¹⁷⁷Lu, ¹⁸⁶Re,        and/or ⁶⁷Ga.    -   48. A method of embodiment 45, wherein the radionuclide includes        ⁸⁹Zr, ²²⁵Ac, and/or ²²⁷Th.    -   49. A method of any one of embodiments 45-48, wherein the metal        includes a daughter isotope of a radionuclide.    -   50. A method of embodiment 49, wherein the daughter isotope of        the radionuclide includes ⁸⁹Y, ¹⁸O, ²²¹Fr, ²¹³Bi, and/or ²⁰⁹Pb.    -   51. A method of any of embodiments 1-50, wherein the treating        provides imaging to aid in diagnosis; to locate a position for a        therapeutic intervention; to assess the functioning of a body        part; and/or to assess the presence or absence of a condition.    -   52. A method of embodiment 51, wherein the imaging is through        positron emission tomography (PET), single photon emission        computed tomography, radioisotope renography, or scintigraphy    -   53. A method of any of embodiments 1-52, wherein the treating        reduces cellular proliferation.    -   54. A method of embodiment 53, wherein the cellular        proliferation is due to cancer, a thyroid disease, a blood        disorder, and/or restenosis.    -   55. A method of embodiment 54, wherein the cancer is adrenal        cancer, bladder cancer, blood cancer, bone cancer, brain cancer,        breast cancer, carcinoma, cervical cancer, colon cancer,        colorectal cancer, corpus uterine cancer, ear, nose and throat        (ENT) cancer, endometrial cancer, esophageal cancer,        gastrointestinal cancer, head and neck cancer, Hodgkin's disease        cancer, intestinal cancer, kidney cancer, larynx cancer,        leukemia, liver cancer, lymph node cancer, lymphoma, lung        cancer, melanoma, mesothelioma, myeloma, nasopharynx cancer,        neuroblastoma, non-Hodgkin's lymphoma, oral cancer, ovarian        cancer, pancreatic cancer, penile cancer, pharynx cancer,        prostate cancer, rectal cancer, sarcomcancer, seminomcancer,        skin cancer, stomach cancer, teratomcancer, testicular cancer,        thyroid cancer, uterine cancer, vaginal cancer, vascular tumor        cancer, and/or cancer from metastases thereof.    -   56. A method of embodiment 54, wherein the thyroid disease is        hyperthyroidism or thyrotoxicosis.    -   57. A method of embodiment 54, wherein the blood disorder is        Polycythemia vera.    -   58. A method of embodiment 54, wherein the restenosis follows        balloon angioplasty and/or stent placement.    -   59. A composition including a mutated siderocalin with position        54 modified from threonine to cysteine, position 68 modified        from serine to cysteine, or both position 54 modified from        threonine to cysteine and position 68 modified from serine to        cysteine.    -   60. A composition of embodiment 59, wherein the mutated        siderocalin is bound to any of the structures included in        embodiments 1-42 at least at position 54, position 68, or both        position 54 and position 68.    -   61. A method of treating a subject in need thereof comprising        administering a therapeutically effective amount of a        composition of embodiments 59 or 60 to the subject thereby        treating the subject.    -   62. A method of synthesizing a siderophore including at least        one SH substituent using dichlorodiphenylmethane.    -   63. A kit including one or more compositions including the        structure included in any of embodiments 1-42, one or more        siderocalins, and/or one or more metals.    -   64. A kit of embodiment 54, wherein the one or more siderocalins        include any of SEQ ID NOs. 1-25.    -   65. A kit of embodiment 54 or 55, wherein the metal is a        radionuclide.    -   66. A kit of embodiment 56 wherein the metal is a radionuclide        of any of embodiments 46-50.    -   67. A method including performing imaging and treatment of a        condition in a same subject using the same combination of a        siderocalin, a chelator, and a metal in the imaging and the        treatment of the subject.    -   68. A method of embodiment 67, wherein the chelator comprises        the structure included in any of embodiments 1-42.    -   69. A method of embodiment 67 or 68, wherein the siderocalin        includes one of SEQ ID NOs. 1-25.    -   70. A method of embodiment 67 or 68 wherein the siderocalin is a        mutated siderocalin with position 54 modified from threonine to        cysteine, position 68 modified from serine to cysteine, or both        position 54 modified from threonine to cysteine and position 68        modified from serine to cysteine.    -   71. A method of embodiment 70, wherein the mutated siderocalin        is bound to the chelator at least at position 54, position 68,        or both position 54 and position 68.    -   72. A method of any of embodiments 67-71, wherein the metal is a        radionuclide.    -   73. A method of any of embodiments 67-71, wherein the metal is a        radionuclide of embodiment 46.    -   74. A method of any of embodiments 67-71 wherein the metal is a        radionuclide selected from ⁹⁰Y, ⁶⁷Cu, ²¹³Bi, ²¹²Bi, ¹⁸⁶Re, ⁶⁷Cu        ⁹⁰Y, ²¹³Bi, ¹⁷⁷Lu, ¹⁸⁶Re, and ⁶⁷Ga.    -   75. A method of any of embodiments 67-71 wherein the metal is a        radionuclide selected from ⁸⁹Zr, ²²⁵Ac, and ²²⁷Th.    -   76. A composition having a structure including:

wherein:(i) A1, A2, A3, and A4, individually, include a CAM group, a 1,2-HOPOgroup, or a HA group;(ii) B31, B2, B3, and B4, individually, include an amide group or anamine group;(iii) at least one of C1, C2, C3, C4, C5, or C6, individually, includeSH;(iv) at least another one of C1, C2, C3, C4, C5, or C6 is optional and,individually, includes C(═O)OH or NH₂;(v) at least one of L1, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11, L12,or L13, individually, include H, an alkyl group having no greater than10 carbon atoms, an alkylamino group having no greater than 10 carbonatoms and no greater than 2 nitrogen atoms; an alkyl ether group havingno greater than 10 carbon atoms, a hydroxy ester group, or an alkylester group having no greater than 10 carbon atoms; and(vi) at least one of L1, L5, L6, L7, L8, L9, L10, L11, L12, or L13 isoptional.

-   -   77. A composition of embodiment 76, wherein at least another one        of L2, L3, or L4, individually, include an amine group or an        amide group.    -   78. A composition of embodiment 76 or embodiment 77, wherein L1,        C1, L7, C2, L9, C3, 11, C4, and L13, C5 are absent, L5 includes        an unsubstituted alkyl group having no greater than 5 carbon        atoms, and C6 includes SH, C(═O)OH, or NH₂.    -   79. A composition of embodiment 78, wherein L2, L3, L4, L6, L8,        L10, and L12, individually, include an unsubstituted alkyl group        having no greater than 5 carbon atoms.    -   80. A composition of embodiment 79, wherein A1 includes a CAM        group or a HOPO group; A2 includes a HA group, A3 includes a HA        group, and A4 includes a CAM group, a HOPO group, or a HA group.    -   81. A composition of any one of embodiments 76-80, wherein at        least one of L2, L3, or L4, individually, include an alkylamino        group.    -   82. A composition of embodiment 76, wherein 1, B2, and B3,        individually, include an amide group and B4 includes an amino        group, L2 and L3 include an amino group, and L4 includes an alky        group having no greater than 5 carbon atoms    -   83. A composition of embodiment 82, wherein:        C1, C2, C3, C4, C5, L1, A1, A2, A3, L1, L6, L7, L8, L9, L10,        L11, L12, and L13 are absent,        A4 includes a CAM group, a HOPO group, or a HA group; and        L5 includes an alkyl group having no greater than 5 carbon        atoms.    -   84. A composition of embodiment 76, wherein 1, B2, and B3,        individually, include an amide group and B4 includes an amide        group, L2 and L3, individually, include an amino group, and L4        includes an alky group having no greater than 5 carbon atoms.    -   85. A composition of embodiment 84, wherein C1, C2, C3, C4, C5,        A1, A2, A3, L1, L6, L7, L8, L9, L10, 11, and L13 are absent, L12        includes an amino group, L5 includes an ether group having no        greater than 10 carbon atoms, and A4 includes a CAM group, a        HOPO group, or a HA group.    -   86. A composition of embodiment 85, wherein C1, C2, C5, C6, L1,        L2, L3, L4, L5, L7, L13, B2, and B4 are absent, 1 and B3,        individually, include an amide group, L6, L8, L10, and L12,        individually, include an amino group, A1, A2, A3, and A4,        individually, include a CAM group, a HOPO group, or a HA group,        L9 and 11, individually, include an alkyl group having no        greater than 5 carbon atoms.    -   87. A composition, including a structure:

wherein:at least one of R₁, R₂, R₃, R₄, and R₅, individually, include a CAMgroup, a HA group, or a 1,2-HOPO group;at least another one of R₁, R₂, R₃, R₄, and R₅, individually, include Hor an alkyl group having from 1 to 10 carbon atoms;R₆ includes an alkyl group having from 1 to 10 carbon atoms andsubstituted by SH;m can be from 1 to 6;n can be from 1 to 6;o can be from 1 to 6.

-   -   88. A composition of embodiment 87, including a structure:

wherein:at least one of R₁, R₃, R₄, or R₅ R₁, R₂, R₃, R₄, and R₅, individually,include a CAM group, a HA group, or a 1,2-HOPO group;optionally, another one of R₁, R₃, R₄, or R₅ R₁, R₂, R₃, R₄, and R₅,individually, include H or an alkyl group having from 1 to 10 carbonatoms;R₂ includes H or an alkyl group including from 1 to 5 carbon atoms;R₇ includes SH; andp is from 0 to 4.

-   -   89. A composition of embodiment 88, wherein:        R₁ includes a CAM group or a 1,2-HOPO group;        R₃ and R₄, individually, include a HA group; and        R₅ includes a CAM group, a 1,2-HOPO group, or a HA group.    -   90. A composition of embodiment 87, including a structure:

wherein:R₇ includes SH;R₂, R₈, and R₉, individually, include H, OH, or an alkyl group includingfrom 1 to 5 carbon atoms; andp is from 0 to 4.

-   -   91. A composition of embodiment 87, including a structure:

wherein:R₇ includes SH;R₂, R₈, and R₉, individually, include H, OH, or an alkyl group includingfrom 1 to 5 carbon atoms; andp is from 0 to 4.

-   -   92. A composition of embodiment 87, including a structure:

wherein:R₇ includes SH;R₂, R₈, R₉, and R₁₀, individually, include H, OH, or an alkyl groupincluding from 1 to 5 carbon atoms; andp is from 0 to 4.

-   -   93. A composition of embodiment 87, including a structure:

wherein:R₇ includes SH;R₂, R₈, and R₉, individually, include H, OH, or an alkyl group includingfrom 1 to 5 carbon atoms; andp is from 0 to 4.

-   -   94. A composition of embodiment 87, including a structure:

wherein:R₇ includes SH;R₂, R₈, and R₉, individually, include H, OH, or an alkyl group includingfrom 1 to 5 carbon atoms; andp is from 0 to 4.

-   -   95. A composition of embodiment 87, including a structure:

wherein:R₇ includes SH;R₂, R₈, R₉, and R₁₀, individually, include H, OH, or an alkyl groupincluding from 1 to 5 carbon atoms; andp is from 0 to 4.

-   -   96. A composition, including a structure:

wherein:at least one of R₁₁, R₁₂, R₁₃, or R₁₅, individually, include a CAMgroup, a HA group, or a 1,2-HOPO group;optionally, at least another one of R₁₁, R₁₂, R₁₃, or R₁₅, individually,include H, OH, or an alkyl group having from 1 to 10 carbon atoms;R₁₇ includes SH;R₂, R₁₄, and R₁₆, individually, include H, OH, or an alkyl group havingfrom 1 to 10 carbon atoms; andr can be from 0 to 6.

-   -   97. A composition of embodiment 96, wherein:        R₁₁ includes a CAM group or a 1,2-HOPO group;        R₁₂ and R₁₅, individually, include a HA group; and        R₁₃ includes a CAM group, a 1,2-HOPO group, or a HA group.    -   98. A composition of embodiment 96, including a structure:

wherein:R₂, R₁₄, R₁₆, R₁₈, and R₁₉, individually, include H, OH, or an alkylgroup having from 1 to 10 carbon atoms; andr can be from 0 to 4.

-   -   99. A composition of embodiment 96, including a structure:

whereinR₂, R₁₄, R₁₆, R₁₈, and R₁₉, individually, include H, OH, or an alkylgroup having from 1 to 10 carbon atoms; andr is from 0 to 4.

-   -   100. A composition of embodiment 96, including a structure:

wherein:R₂, R₁₄, R₁₆, R₁₈, R₁₉, and R₂₀, individually, include H, OH, or analkyl group having from 1 to 10 carbon atoms; andr can be from 0 to 4.

-   -   101. A composition of embodiment 96, including a structure:

wherein:R₂, R₁₄, R₁₆, R₁₈, R₁₉, and R₂₀, individually, include H, OH, or analkyl group having from 1 to 10 carbon atoms; andr can be from 0 to 4.

-   -   102. A composition of embodiment 96, including a structure:

wherein:R₂, R₁₄, R₁₆, R₁₈, and R₁₉, individually, include H, OH, or an alkylgroup having from 1 to 10 carbon atoms; andr is from 0 to 4.

-   -   103. A composition of embodiment 96, including a structure:

wherein:R₂, R₁₄, R₁₆, R₁₈, and R₁₉, individually, include H, OH, or an alkylgroup having from 1 to 10 carbon atoms;r is from 0 to 4.

-   -   104. A composition, including a structure:

wherein:R₂₁ and R₂₂, individually, include H, OH, or an alkyl group having from1 to 10 carbon atoms; R₂₃ includes H, OH, an alkyl group having from 1to 10 carbon atoms, or (CH₂)_(e)R_(a), where R_(a) is SH;R₂₄ includes a substituent having a CAM group, a 1,2-HOPO group, or a HAgroup;a, b, and c, individually, are from 1 to 10;d is from 1 to 4; ande is from 1 to 10.

-   -   105. A composition of embodiment 104, wherein R₂₄ includes a        substituent having SH, C(═O)OH, or NH₂.    -   106. A composition of embodiment 104, including a structure:

wherein:R₂₅, R₂₆, and R₂₇, individually, include H, OH, or an alkyl group havingfrom 1 to 10 carbon atoms;R₂₈ includes SH; ands is from 0 to 4.

-   -   107. A composition of embodiment 104, including a structure:

wherein:R₂₅, R₂₆, R₂₇, and R₃₀, individually, include H, OH, or an alkyl grouphaving from 1 to 10 carbon atoms;at least one of R₂₈ or R₂₉, individually, includes SH and the other ofR₂₈ or R₂₉ includes H, an alkyl group having from 1 to 5 carbon atoms,SH, NH₂, or C(═O)OH;s is from 0 to 4; andt is from 0 to 4.

-   -   108. A composition of embodiment 104, including a structure:

wherein:R₂₅, R₂₆, R₂₇, and R₃₀, individually, include H, OH, or an alkyl grouphaving from 1 to 10 carbon atoms;at least one of R₂₈ or R₂₉, individually, includes SH and the other ofR₂₈ or R₂₉ includes H, an alkyl group having from 1 to 5 carbon atoms,SH, NH₂, or C(═O)OH;s is from 0 to 4; andt is from 0 to 4.

-   -   109. A composition of embodiment 104, including a structure:

wherein:R₂₅, R₂₆, R₂₇, R₃₀, and R₃₁, individually, include H, OH, or an alkylgroup having from 1 to 10 carbon atoms;at least one of R₂₈ or R₂₉, individually, includes SH and the other ofR₂₈ or R₂₉ includes H, an alkyl group having from 1 to 5 carbon atoms,SH, NH₂, or C(═O)OH;s is from 0 to 4; andt is from 0 to 4.

-   -   110. A composition of embodiment 104, including a structure:

whereinR₂₅, R₂₆, and R₂₇, individually, include H, OH, or an alkyl group havingfrom 1 to 10 carbon atoms;R₂₈ includes SH; ands is from 0 to 4.

-   -   111. A composition of embodiment 104, including a structure:

wherein:R₂₅, R₂₆, R₂₇, and R₃₂, individually, include H, OH, or an alkyl grouphaving from 1 to 10 carbon atoms;R₂₈ includes SH; ands is from 0 to 4.

-   -   112. A composition, including a structure:

wherein:A, B, C, and D, individually, include one or more amide groups, one ormore amine groups, or an alkyl group having from 1 to 10 carbon atoms;R₃₃, R₃₄, R₃₅, and R₃₆, individually, include a CAM group, a 1,2-HOPOgroup, or a HA group and at least one of R₃₃, R₃₄, R₃₅, or R₃₆ aresubstituted by SH; andg, h, i, and j, individually, are from 1 to 10.

-   -   113. A composition of embodiment 112, including a structure:

wherein:at least one of R₃₇ or R₄₂, individually, includes SH and the other ofR₃₇ or R₄₂ includes H, an alkyl group having from 1 to 5 carbon atoms,SH, C(═O)OH, or NH₂;R₃₈, R₃₉, R₄₀, and R₄₁, individually, include H, OH, or an alkyl grouphaving from 1 to 5 carbon atoms; andu and v, individually, are from 0 to 5.

-   -   114. A composition of embodiment 112, including a structure:

wherein:at least one of R₃₇ or R₄₂, individually, includes SH and the other ofR₃₇ or R₄₂ includes H, an alkyl group having from 1 to 5 carbon atoms,SH, C(═O)OH, or NH₂;R₃₈, R₃₉, R₄₀, and R₄₁, individually, include H, OH, or an alkyl grouphaving from 1 to 5 carbon atoms; andu and v, individually, are from 0 to 5.

-   -   115. A composition of embodiment 112, including a structure:

wherein:at least one of R₃₇ or R₄₂, individually, includes SH and the other ofR₃₇ or R₄₂ includes H, an alkyl group having from 1 to 5 carbon atoms,SH, C(═O)OH, or NH₂;R₃₈, R₃₉, R₄₀, and R₄₁, individually, include H, OH, or an alkyl grouphaving from 1 to 5 carbon atoms; andu and v, individually, are from 0 to 5.

-   -   116. A method of separating metal ions, comprising:        -   contacting a liquid comprising a plurality of metal ions            with a composition having the structure included in any one            of embodiments 1-42, under conditions sufficient to form a            metal ion-composition complex comprising a metal ion of the            plurality of metal ions; and separating a first fraction of            the mixture enriched for the metal ion-composition complex            from a second fraction depleted for the metal            ion-composition complex, wherein the first fraction is            enriched for a first metal ion that has a charge that is            different from a charge of a second metal ion enriched in            the second fraction.    -   117. The method of embodiment 116, wherein the separating is        based on molecular weight.    -   118. The method of embodiment 116 or 117, wherein the separating        comprises size-exclusion chromatography or affinity        chromatography.    -   119. The method of any one of embodiments 116-118, wherein:        -   a) the first fraction is enriched for a trivalent metal ion            or a divalent metal ion, and the second fraction is enriched            for a tetravalent metal ion;        -   b) the first fraction is enriched for a metal ion selected            from the group consisting of: actinides, lanthanides, Ac³⁺,            Sc³⁺, Y³⁺, and In³⁺;        -   c) the second fraction is enriched for a metal ion selected            from the group consisting of: Pu⁴⁺, Np⁴⁺, Th⁴⁺, Zr⁴⁺, Sn⁴⁺,            Ce⁴⁺, and Bk⁴⁺;        -   d) the first fraction is enriched for a metal ion selected            from the group consisting of Am³⁺, Cm³⁺, Bk³⁺ and Cf³⁺, and            the second fraction comprises Pu⁴⁺;        -   e) the first fraction is enriched for Ac³⁺ and/or Sc³⁺, and            the second fraction is enriched for Th⁴⁺;        -   f) the first fraction is enriched for Eu³⁺ or Y³⁺, and the            second fraction is enriched for Zr⁴⁺;        -   g) the first fraction is enriched for In³⁺, and the second            fraction is enriched for Sn⁴⁺;        -   h) the first fraction is enriched for a lanthanide, and the            second fraction is enriched for Ce⁴⁺;        -   i) the first fraction is enriched for Tm³⁺;        -   j) the first fraction is enriched for an actinide, and the            second fraction is enriched for Bk⁴⁺; or        -   k) the first fraction is enriched for a metal ion selected            from the group consisting of Am³⁺, Cm³⁺, and Cf³⁺.    -   120. A method of preparing Bk⁴⁺ from a mixture, comprising:        -   contacting a first mixture comprising Bk⁴+ and a trivalent            metal ion with a composition having a structure included in            of any one of embodiments 1-42 under conditions sufficient            to form a complex comprising the trivalent metal ion and the            composition having the structure included in any one of            embodiments 1-42;        -   separating the complex from the first mixture to generate a            second mixture depleted for the trivalent metal ion; and        -   chromatographically isolating the Bk⁴⁺ in the second            mixture.    -   121. The method of embodiment 120, wherein:        -   a) the first mixture further comprises one or more actinides            selected from the group consisting of: Cm³⁺, Am³⁺, Cf³⁺,            Th⁴⁺, Np⁴⁺, Pu⁴⁺, and Ce⁴⁺;        -   b) the first mixture is prepared by neutron irradiation of            Pu, Am or Cm;        -   c) the composition comprises a hydroxypyridonate ligand; or        -   d) the composition comprises 3,4,3-LI(1,2-HOPO)    -   122. A method of reclaiming an actinide from a sample, the        method comprising:        -   obtaining an aqueous sample comprising, or suspected of            comprising, an actinide;        -   contacting the sample with a composition having a structure            included in any one of embodiments 1-42 to generate a            mixture under conditions sufficient to form a complex            comprising the actinide and the composition; and        -   separating the complex from the mixture.    -   123. The method of embodiment 122, wherein:        -   a) the actinide comprises Am³⁺ and/or Cm³⁺;        -   b) the sample is derived from a river, ocean, lake, soil, or            industrial run off;        -   c) the sample is an industrial sample;        -   d) the composition comprises a hydroxypyridonate ligand; or        -   e) the composition comprises 3,4,3-LI(1,2-HOPO).

Experimental Examples. Example 1. Tightening the Recognition ofTetravalent Zr and Th Complexes by the Siderophore-Binding MammalianProtein Siderocalin for Theranostic Applications.

Introduction. Targeted alpha therapy (TAT), or radioimmunotherapy thatuses α-particle emitting nuclides, is a promising treatment for smallmetastatic tumors and other localized diseases. Owing to α-particles'short path length, much of the decay energy may be deposited into targetareas while mitigating damage to surrounding tissue (Mulford, et al.,Journal of Nuclear Medicine, 46 (1 suppl), 199S-204S, 2005). A number ofradionuclides that emit single α particles, including ²¹³Bi and ²¹²Pb,are currently under investigation (Kim & Brechbiel, Tumor Biol., 33:573-590, 2012). A growing subset of the field includes in vivoα-generator radionuclides ²²⁵Ac, ²²³Ra, and ²²⁷Th, isotopes that emitmultiple α particles in their decay chains and dramatically increase thepotential delivered dose (McDevitt, et al., Science, 2001, 294 (5546):1537-1540). This principle was recently exploited in the development ofAlpharadin, ²²³RaCl₂, a drug for bone metastases (Liepe & Alpharadin,Curr Opin Investig Drugs, 2009, 10 (12): 1346-1358). While Alpharadinrelies on the natural propensity of ²²³Ra for bone, other specificallytargeted α-radiation delivery strategies use constructs formed with achelating agent to complex metallic α-emitters and a cancer sitetargeting vector (i.e., targeting ligand) (Kim & Brechbiel, Tumor Biol.,33: 573-590, 2012). Though sound in theory, these designs have been slowto appear in the clinic, with only scarce examples of promisingα-generator immunoconjugates, such as the lintuzumab conjugate²²⁵Ac-HuM195 for myeloid leukemia treatment (Ravandi, et al., Blood,2013, 122 (21): 1460-1460) and ²²⁷Th-DOTA-trastuzumab for treating HER-2positive breast and ovarian cancer (Heyerdahl, et al., PloS one, 2012, 7(8): e42345). There are many reasons for this slow development;inadequate chelation being one of the major limitations, together withpoor retention of the α-emitters and their respective daughter productsat the target site.

The ²²⁵Ac and ²²⁷Th radioisotopes are members of the actinide (An)series of elements. They display high coordination numbers and are bestchelated by high denticity chelators that contain hard donor atoms, suchas the multidentate hydroxypyridinone-based (HOPO) compounds, workhorsechelators for in vivo actinide decorporation (Bunin, et al., Rad. Res.2013, 179: 171-182). The chelator 3,4,3-LI(1,2-HOPO) is an octadentate,tetraprotic compound including 4 bidentate 1,2-HOPO metal binding unitsattached onto a spermine (“3,4,3-LI”) scaffold (FIG. 1), which wasrecently modified to enable monoclonal antibody attachment and form abioconjugate chelator that displayed great properties for positronemission tomography (PET) when bound to ⁸⁹Zr (Deri, et al., BioconjugateChemistry, 2015, 26 (12): 2579-2591; Deri, et al., J. Med. Chem., 2014,57 (11): 4849-4860). Although successful, such modifications necessitateextensive synthetic procedures with fairly low yields (Deri, et al.,Bioconjugate Chemistry, 2015, 26 (12): 2579-2591), which promptedinvestigation of alternate routes to link therapeutic and imagingradionuclides to targeting ligands.

The mammalian protein, the siderophore-binding siderocalin (Scn), bindslanthanide (Ln) and An ions pre-complexed with a suitable ligand insolution with very high affinity (Allred, et al., PNAS, 2015, 112 (33):10342-10347). Although the ferric complex of the hexadentate catecholateenterobactin ([Fe^(III)(Ent)]³⁻) is Scn's native ligand (Clifton, etal., Biometals, 2009, 22 (4): 557-564), Scn's sterically hinderedbinding pocket was shown to bind Ln and An complexes of Ent (FIG. 1)(Allred, et al., PNAS, 2015, 112 (33): 10342-10347). More surprisingly,the protein could also accommodate the much stronger Ln and An complexesformed with the octadentate synthetic analog 3,4,3-LI(1,2-HOPO) (Allred,et al., PNAS, 2015, 112 (33): 10342-10347). Building on this work, onecan envision using the Scn-ligand-metal system as a radionuclide binderfor TAT as well as a reporter ligand for concurrent diagnostics. Theadvantage of using a protein-mediated binding system is two-fold:potentially tighter binding to nuclides of interest and retention ofdaughter products within the macromolecular construct, as well as easierconjugation to targeting ligands by using well-established biochemicalmethods. The aforementioned ²²⁵Ac³⁺ and ²²⁷Th⁴⁺ show promise inradioimmunotherapy, while ⁸⁹Zr⁴⁺ is useful as a PET tracer; all of thesemetals may be captured by the Scn-ligand system. Formed upondeprotonotation of the 1,2-HOPO units (FIG. 1), overall negative3,4,3-LI(1,2-HOPO) complexes of Ln³⁺ and An³⁺ are tightly bound by Scn,but formally neutral 3,4,3-LI(1,2-HOPO) complexes of 4⁺ metals interactweakly (Allred, et al., PNAS, 2015, 112 (33): 10342-10347). Thedifference in binding originates from insufficient electrostaticinteraction between the metal-chelator complex and Scn. In order torestore complex binding by Scn, a ligand of high denticity that bindsboth 3⁺ and 4⁺ metals, forms overall negatively charged complexes, anddoes not cause steric clashes in Scn's tight binding pocket should beused. This Example aimed to address the inability of theScn-3,4,3-LI(1,2-HOPO) system to bind 4⁺ ions by exploring more suitablechelators.

Materials and Methods. Caution. ²³²Th, ²³⁸Pu, ²⁴²Pu, ²⁴³Am, and ²⁴⁸Cmare hazardous radionuclides with high specific activities that shouldonly be manipulated in specifically designated facilities in accordancewith appropriate safety controls.

General Considerations. Chemicals were obtained from commercialsuppliers and were used as received. The siderophore Ent was provided byProf. K. N. Raymond (Department of Chemistry, University of Californiaat Berkeley). The LnCl₃.nH₂O lanthanide salts utilized were of thehighest purity available (>99.9%). Stock solutions of ²³²Th(IV) andZr(IV) were prepared from ²³²ThCl₄.H₂O (Baker & Adamson, ACS grade) andZrCl₄ (Sigma-Aldrich, 99.99%), respectively. A stock of ²³⁸Pu(IV) waspurchased as ²³⁸Pu(NO₃)₄ in 4 M HNO₃ from Eckert & Ziegler (lot 118521).²⁴²Pu was received from Oak Ridge National Laboratory as PuO2 (lotPu-242-327 A, 99.93 wt % of metal ²⁴²Pu) and a stock solution of²⁴²Pu(IV) was prepared as described previously (Ravandi, et al., Blood,2013, 122 (21): 1460-1460). The ²⁴²Pu isotope was used for in vitrobinding experiments whereas ²³⁸Pu was used in biodistribution studies.Aliquots of acidified stocks of carrier-free ²⁴³Am and ²⁴⁸Cm (95.78%²⁴⁸Cm, 4.12% ²⁴⁶Cm, 0.06% ²⁴⁵Cm, 0.02% ²⁴⁴Cm/²⁴⁷Cm isotopic distributionby atom percentage) from Lawrence Berkeley National Laboratory wereused. All solutions were prepared using deionized water purified by aMillipore Milli-Q reverse osmosis cartridge system, and special care wastaken to adjust the pH with concentrated HCl, H₂SO₄, KOH, or NaOH whenneeded. Measurements were conducted at room temperature unless otherwisenoted. ¹H NMR spectra were recorded on Bruker instruments; ¹³C NMRspectra were recorded on Bruker instruments with tetramethylsilane as aninternal reference. SilicaFlash G60 (particle size 60-200 μm) was usedfor flash column chromatography. LC-MS was performed on an Agilent LC/MSsystem including an Agilent 1200 binary LC pump, atemperature-controlled autosampler, a PDA UV detector, and a 6530Accurate Mass Q-TOF mass spectrometer (Wilmington, Del., USA). The massspectrometer was equipped with a JetStream® ESI probe operating atatmospheric pressure. The ESI source parameter settings were: mass rangem/z 100-1000, gas temperature 350° C., gas flow 10 L/min, nebulizer 50psi, sheath gas temperature 400° C., sheath gas flow 12 L/min, capillaryvoltage (Vcap) 3500 V, nozzle voltage 500 V, fragmentor 200 V, skimmer65 V, octopole RF (OCT 1 RF Vpp) 750 V. Reverse phase preparatory HPLCwas performed on a Varian ProStar system with a Vydac C18 column.High-resolution mass spectra were acquired using a Waters Xevo G2 QTofmass spectrometer. Absorption spectra were recorded on a Varian Cary G5double beam absorption spectrometer or a NanoDrop 2000C, using quartzcells of 10 and 2 mm path lengths, respectively.

Methyl 2,3-dihydroxybenzoate (2). A stirred suspension of 1 (8.06 g,52.3 mmol) in 100 mL of MeOH was treated with 2.00 ml of concentratedsulfuric acid. The suspension warmed and clarified 2 minutes after theaddition. The reaction was equipped with a reflux condenser and washeated to 65° C. overnight. The next morning the conversion was verifiedby LC-MS and the volatiles were removed under reduced pressure. Thecrude was partitioned between H₂O (100 mL) and ethyl acetate (100 mL)and the aqueous layer was extracted with ethyl acetate (3×50 mL). Theorganic extracts were combined, dried over MgSO₄, and concentrated underreduced pressure. The crude was passed through a plug of silica using10% ethyl acetate in hexanes as eluent. The eluent was concentratedunder reduced pressure and dried under high vacuum for 2 hours to yield2 (7.66 g, 45.6 mmol, 88%) as a white solid, the spectral properties ofwhich matched previous reports (Weitl, et al., J. Am. Chem. Soc., 1980,102 (7): 2289-2293).

Methyl 2,2-diphenylbenzo[d][1,3]dioxole-4-carboxylate (3). Precursor 2(5.00 g, 29.7 mmol) was mixed with dichlorodiphenylmethane (8.56 mL,44.6 mol) under an argon atmosphere; the resulting suspension wasstirred and heated to 160° C. for 1 hour. The mixture was allowed tocool to room temperature and was diluted with 100 mL of ethyl acetate.The solution was washed with sat. NaHCO₃ (30 mL), brine (30 mL), driedover MgSO₄, and then concentrated under reduced pressure. The ensuinggreyish oil was dissolved in 30 mL of hot MeOH (65° C.) and was slowlycooled to 5° C., which resulted in the formation of white crystals. Thecrystals were a mixture of 3 and benzophenone that could not be easilyseparated; the crude product was used as is for the subsequent step.

2,2-diphenylbenzo[d][1,3]dioxole-4-carboxylic acid (4). The mixture fromthe previous step was dissolved in 100 mL of THF and was treated with100 mL of 0.9 M LiOH. The emulsion was rapidly stirred and heated toreflux for 5 hours. Conversion was verified by LC-MS and the reactionwas cooled to room temperature. The solution was neutralized with 10%v/v aqueous acetic acid and was extracted with ethyl acetate (3×50 mL).The organic extracts were combined, dried over MgSO₄, and concentratedunder reduced pressure. The crude was chromatographed using 25% ethylacetate in hexanes as eluent. Volatiles were then removed under reducedpressure followed by high vacuum to yield 4 (7.6 g, 24.06 mmol, 81% over2 steps) as a white solid, the spectral properties of which matchedprevious reports (Weitl, et al., J. Am. Chem. Soc., 1980, 102 (7):2289-2293).

3,4,3-LI(2,2-diphenylbenzo[d][1,3]-2,3-catecholamide) (5). Precursor 4(746 mg, 2.33 mmol) was suspended in 10 mL of dry toluene under an argonatmosphere and was treated with oxalyl chloride (220 μL, 2.55 mmol).Catalytic N,N-dimethylformamide was added and the suspension was heatedto 40° C. The solution was stirred until the evolution of gas ceased andwas concentrated on the manifold vacuum at the same temperature. Theresulting brown oil was dissolved in 10 mL of dry THF. In a separatecontainer a solution of spermine (118 mg, 0.583 mmol), triethylamine(356 μL, 2.56 mmol), and THF (5 mL) was prepared. The solutions werecombined and heated to 50° C. overnight in a sealed flask. The followingday the reaction was filtered and concentrated under reduced pressure.The resulting crude oil was chromatographed using 3% MeOH in CH₂Cl₂ aseluent. The volatiles were then removed under reduced pressure and driedunder vacuum, yielding 5 as a white foam (641 mg, 0.457 mmol, 78%yield). ¹H NMR (300 MHz, CDCl₃) δ 7.88 (1H, t, J=5.7 Hz), 7.66-7.76 (6H,br t), 7.60 (1H, br s), 7.57 (1H, br s), 7.43-7.53 (10H, br s),7.33-7.40 (4H, br s), 7.19-7.31 (20H, br s), 7.01 (2H, d, J=7.6 Hz),6.91 (4H, dd, J=12.1 Hz, 8.0 Hz), 6.80 (2H, br s), 6.72 (2H, br s), 3.85(4H, br s), 3.43 (2H br s), 3.21 (2H, br s), 3.06 (1H, br s), 2.96 (1H,br s), 2.80 (2H, br s), 1.81 (4H, br s), 1.54 (1H, br s), 1.43 (1H, brs), 1.19 (1H, br s), 0.89 (2H, br s). ¹³C NMR (75 MHz, CDCl₃) δ 167.5,163.7, 147.3, 147.1, 145.0, 142.8, 139.7, 139.4, 138.9, 129.7, 129.2,128.4, 128.3, 126.4, 126.3, 126.1, 126.0, 122.3, 122.2, 121.7, 120.4,118.4, 118.1, 116.0, 111.8, 111.4, 111.3, 109.4, 47.9, 41.8, 36.5, 27.9,25.5 (FIG. 3).

3,4,3-LI(CAM) (6). The protected chelator 5 (411 mg, 2.93×10⁻⁴ mol) wasdissolved in a mixture of 5 mL acetic acid, 0.5 mL H₂O, and 0.1 mLconcentrated HCl. The solution was stirred in a sealed container at 60°C. overnight. The next day the conversion was confirmed by LC-MS and thevolatiles were removed under vacuum. A portion of the crude was purifiedusing reverse-phase prep-HPLC using at 10→50% MeOH in H₂O+0.1%trifluoroacetic acid as eluent. The solvent was removed on a Genevaccentrifugal evaporator followed by lyophilization of residual H₂O. CAMwas obtained as a pure white powder (90% yield). ¹H NMR (600 MHz,DMSO-d₆) δ 12.82 (1H, br s), 12.69 (1H, br s), 9.52 (2H, br s), 9.11(2H, br s), 8.78 (1H, br s), 8.60 (3H, br s), 7.26 (1H, br s), 7.12 (1H,br s), 6.90 (2H, br s), 6.77 (1H, br s), 6.66 (4H, br s), 6.56 (2H, brs), 6.44 (1H, br s), 2.88-3.52 (12H, overlapping aliphatic signals),1.16-1.83 (8H, overlapping aliphatic signals); ¹³C NMR (125 MHz,MeOD-d₄) δ 172.9, 171.5, 150.4, 147.3, 146.6, 125.6, 125.4, 121.0,119.6, 119.1, 118.8, 118.6, 116.9, 116.6, 47.7, 44.9, 43.2, 37.8, 37.5,29.3, 28.2, 26.5, 25.5 (FIG. 3). MS-ESI (m/z) [M+H] Calcd forC₃₈H₄₃N₄O₁₂, 747.2878; found 747.2922 and [M−H] Calcd. for C₃₈H₄₁N₄O₁₂,745.2721; found 745.2774 (FIG. 5).

Another pathway for synthesizing a chelator that includes a carboxylgroup for binding with another compound, such as a protein or a dye, caninclude:

Metal, Chelator, and Protein Working Solutions. The trivalent lanthanideLn(III) working stock solutions were prepared in standardized 0.1 M HCl.A Zr(IV) stock solution was prepared by dissolving ZrCl₄ in 3.0 M H₂SO₄,to prevent hydrolysis. The metal salt ZrCl₄ was handled and stored in aglovebox kept under inert atmosphere. The Zr(IV) stock solution wasstandardized against EDTA, with xylene orange as the indicator (Welcher,F. J. The analytical uses of ethylenediamine tetraacetic acid; 1958). ATh(IV) stock solution was prepared in 0.1 M H₂SO₄. Stock solutions (4mM) of Ent, and 3,4,3-LI(CAM) were prepared by direct dissolution of aweighed portion of chelator in DMSO and aliquots were removed prior toeach set of experiments. Recombinant human Scn was prepared aspreviously described (Goetz, et al., Molecular cell, 2002, 10 (5):1033-1043).

Solution Thermodynamics. All titrant solutions were degassed by boilingfor 1 h while being purged under Ar. Carbonate-free 0.1 M KOH wasprepared from concentrate (J.T Baker Dilut-It) and was standardized bytitrating against 0.1 M potassium hydrogen phthalate (99.95%, SigmaAldrich). Solutions of 0.1 M HCl were similarly prepared and werestandardized by titrating against TRIS (99.9%, J.T. Baker). The glasselectrode (Metrohm-Micro Combi-response to [H+]) used for the pHmeasurements was calibrated at 25.0° C. and at an ionic strength of 0.1M (KCl) before each potentiometric or spectrophotometric titration. Thecalibration data were analyzed using the program GLEE (Gans &O'Sullivan, Talanta, 2000, 51 (1): 33-37) to refine for the E° andslope. All thermodynamic measurements were conducted at 25.0° C., in 0.1M KCl supporting electrolyte under positive Ar gas pressure. Theautomated titration system was controlled by an 867 pH Module (Metrohm).Two-milliliter Dosino 800 burets (Metrohm) dosed the titrant (0.1 M KOHor 0.1 M HCl) into the thermostated titration vessel (5-90 mL).UV-visible spectra were acquired with an Ocean Optics USB4000-UV-visspectrometer equipped with a TP-300 dip probe (Ocean Optics; path lengthof 10 mm), fiber optics and a DH-2000 light source (deuterium andhalogen lamps). The fully automated titration system and the UV-visspectrophotometer were coordinated by LBNL titration system, a computerprogram developed in house.

Incremental Spectrophotometric Titrations. This method was used todetermine the protonation constants of 3,4,3-LI(CAM) as well as thestability constants of its complexes formed with Eu(III), Zr(IV) and²³²Th(IV). The experimental titration setup is similar to previouslydescribed systems (Sturzbecher-Hoehne, et al., Radiochimica Acta., 2013,101 (6): 359-366). For the 3,4,3-LI(CAM) protonation (andEu(III)-3,4,3-LI(CAM) complexes), titrations were performed with aninitial concentration of 50 μM of 3,4,3-LI(CAM) (and 50 μM of Eu(III))resulting in absorbance values included between 0 and 1.0 throughout thetitration. Typically, 9 mL of a sample containing 3,4,3-LI(CAM) (andEu(III)) and the supporting electrolyte (KCl/HCl) were incrementallyperturbed by addition of 0.025 mL of carbonate-free 0.1 M KOH followedby a time delay of 80 s. Buffering of the solution was ensured by theaddition of 10 mM of HEPES, 10 mM of CHES and 10 mM of MES. Between 130and 250 data points were collected per titration, each data pointincluding a pH measurement and a UV-Vis spectrum (250-450 nm) over thepH range 1.5 to 12.0. All spectra were corrected for dilution beforedata fitting. The entire procedure (electrode calibrate, titration anddata treatment) was performed independently five times for theprotonation constants and four times for the Eu(III)-3,4,3-LI(CAM)complexes. For the Zr(IV) and Th(IV) complexes, titrations wereperformed similarly but in the presence of DTPA to avoid the formationof metal hydroxides at low pH, before the uptake by 3,4,3-LI(CAM). Foreach metal, three titrations were performed independently in thepresence of 1.1 to 40 equivalents of DTPA. Examples of titrations aredisplayed in the Supporting Information (FIGS. 5-7).

Data Treatment. Thermodynamic constants and spectral deconvolution wererefined using the nonlinear least-squares fitting program HypSpec (Gans,et al., Talanta, 1996, 43 (10): 1739-1753). All equilibrium constantswere defined as cumulative formation constants, δ_(mlh) according toEquation (1), where the metal and chelator are designated as M and L,respectively. All metal and chelator concentrations were held atestimated values determined from the volume of standardized stocksolutions. All species formed with 3,4,3-LI(CAM) were considered to havesignificant absorbance to be observed in the UV-vis spectra and weretherefore included in the refinement process. The refinements of theoverall formation constants β included in each case with previouslydetermined chelator protonation constants and the metal hydrolysisproducts, whose equilibrium constants were fixed to the literaturevalues (Smith, et al., NIST standard reference database 46. NISTCritically selected stability constants of metal complexes database ver2004, 2) The speciation diagrams were calculated using the modelingprogram Hyss (Alderighi, et al., Coordination Chemistry Reviews, 1999,184 (1): 311-318). Errors on log δ_(mlh) and pK_(a) values presented inthis Example correspond to the standard deviation observed over the nreplicates (n=3 to 5) of the entire procedure (electrode calibrate,titration and data treatment).

mM+IL+hH⇄[M _(m) L _(l) H _(h)];β_(mlh)=[M _(m) L _(l) H_(h)]/([M]^(m)[L]^(l)[H]^(h))

Fluorescence Quenching Binding Assay. Equimolar amounts of metal andchelator were used to constitute metal-chelator solutions (2 μM, pH 7.4,5% DMSO) in Tris-buffered saline (TBS). Then, a solution of recombinantwild-type Scn (50 nM, 3 mL, 10 μg/mL ubiquitin, TBS pH 7.4, 5% DMSO) wastitrated with the metal-chelator solution. Fluorescence quenching of Scnwas measured after each titrant addition on a HORIBA Jobin Yvon IBHFluoroLog-3 spectrofluorimeter, with 3 nm slit band-pass, using thecharacteristic excitation and emission wavelengths λ_(exc)=280 andλ_(em)=320-360 nm. The intrinsic fluorescence in proteins is generallyattributed to tryptophan residues; two residues W31 and W79 are found inthe proximity of the Scn binding site. Fluorescence values werecorrected for dilution upon addition of titrant. Fluorescence data wereanalyzed by nonlinear regression analysis of fluorescence responseversus chelator concentration using a one-site binding model asdescribed elsewhere. Allred, et al., PNAS, 2015, 112 (33): 10342-10347)The K_(d) values are the results of at least three independenttitrations were determined according to Equation (2). Controlexperiments were performed with [Fe^(III)(Ent)]³⁻ to ensure thestability of the protein under experimental conditions.

Crystallography. For crystallization, 1 mM solutions of equimolarmetal/chelator complexes (prepared as above) were mixed in a 2:1 molarratio with Scn, which was then buffer-exchanged into 25 mM PIPES(pH=7.0), 150 mM NaCl, 1 mM EDTA, and 0.01% w/w NaN₃, and concentratedto 10 mg/ml protein. Diffraction-quality crystals were grown by vapordiffusion from drops containing 1 μl of ternary metal-chelator-proteincomplex plus 1 μl of well solution (50 mM NaCl, 200 mM Li₂SO₄, 100 mMNaOAc (pH=4.3-4.5), 1.2-1.4 M (NH₄)₂SO₄). Crystals were cryo-preservedby transfer to 50 mM NaCl, 200 mM Li₂SO₄, 100 mM NaOAc (pH=4.3-4.5), 1.2M (NH₄)₂SO₄, and 20% v/v glycerol. Diffraction data were collected onbeamline 5.0.2 at the Advanced Light Source (ALS, Berkeley, Calif.).Diffraction data were integrated and scaled with HKL-2000 (Otwinowski &Minor, Processing of X-ray Diffraction Data Collected in OscillationMode. In Methods in Enzymology, Carter, C. W., Jr.; Sweet, R. M., Eds.Academic Press: New York, 1997; Vol. 276, pp 307-326.). Initial phaseswere determined by rigid body positional refinement with Refmac(Murshudov, et al., Acta Crystallogr D Biol Crystallogr, 1997, 53:240-255) using 3FW5.pdb as a starting structure, or molecularreplacement with MolRep (Vagin & Teplyakov, MOLREP: an automated programfor molecular replacement. J. Appl. Cryst., 1997, 30, 1022-1025) using3FW5.pdb as a search model. Structures were refined through iterativerounds of positional refinement using Refmac (Murshudov, et al., ActaCrystallogr D Biol Crystallogr, 1997, 53: 240-255) alternating withmodel building using COOT, 30 followed by a final round of TLSrefinement. 31 Residues or side-chains that did not exhibit clearelectron density in 2F_(obs)-F_(calc) Fourier syntheses when contouredat 0.7σ were removed or truncated to the Cβ atom. The quality of thefinal model was assessed using ProCheck (Laskowski, et al., J. Appl.Cryst., 1993, 26: 283-291) and Molprobity (Davis, et al., Nucleic AcidsRes., 2007, 35: W375-383.). Crystallographic statistics are reported inFIG. 9. Final models have been deposited in the PDB (Berman, et al.,Nucleic Acids Res., 2000, 28 (1), 235-242).

In Vivo Biodistribution Assay. All procedures and protocols used in thepresented in vivo studies were reviewed and approved by theInstitutional Animal Care and Use Committee at Lawrence BerkeleyNational Laboratory and performed in AAALAC accredited facilities. Theanimals used were adult female CD-1 mice (180±7 days old, 40.8±5.8 g).Solutions of ²³⁸Pu complexed by 3,4,3-LI(CAM) and Scn:3,4,3-LI(CAM) wereprepared in situ at molar ratios protein:ligand:²³⁸Pu of 0:100:1 and100:100:1, respectively by mixing and incubating the appropriatequantities of ²³⁸Pu(NO₃)₄, ligand, and protein in phosphate-bufferedsaline (PBS) to reach a ²³⁸Pu concentration of 12 ng L-1. Proteinsolutions were washed thrice with PBS using 10 kDa molecular weightcut-off membrane-based centrifugal filters, and all solutions werefilter-sterilized (0.22 μm) prior to injection. Under isofluraneanesthesia, groups of three normally fed mice were injectedintravenously with 0.2 mL of a complex solution (370 Bq per mouse).After injection of the ²³⁸Pu tracer, mice were weighed, identified, andhoused in groups of three in plastic stock cages lined with a 0.5 cmlayer of highly absorbent low-ash pelleted cellulose bedding (Alpha-dri)for separation of urine and feces. Mice were given water and food adlibitum and euthanized at 4, 24, or 48 h after tracer injection. Allexperiments using ²³⁸Pu tracers were managed as metabolic balancestudies, in which tissues and excreta were analyzed for ²³⁸Pu by liquidscintillation counting on a Perkin Elmer Packard Tri-Carb model B4430.The methods of sample collection, preparation, radioactivitymeasurements, and data reduction have been published previously(Sturzbecher-Hoehne, et al., Dalton Transactions, 2011, 40 (33):8340-8346: Kullgren, et al., Toxicology mechanisms and methods, 2013, 23(1), 18-26; Durbin, et al., Health physics 2000, 78 (5): 511-521). Thosemethods provide quantitative measurements of radioactivity in biologicalsamples; material recoveries averaged 99% of the amount injected inthese experiments.

Results & Discussion. Synthesis of Octadentate Ligand 3,4,3-LI(CAM).Since electrostatic interactions between Scn and Ln/An complexes play akey role in binding, chelators that would form overall negativecomplexes with both 3+ and 4+ metals were explored. Although Scnexhibits a broad, degenerate recognition mechanism for nativesiderophores, previous studies probing the extent of Scn binding tosynthetic siderophore analogs showed that the Scn binding site allowsonly limited changes to its ligands (Abergel, et al., J Am Chem Soc,2006, 128 (34): 10998-10999; Holmes, et al., Structure, 2005, 13 (1)29-41). Thus, the simplest way to correct the binding would be to employchelators with similar structural features. Occam's razor was followedby using 3,4,3-LI(CAM), a known compound first prepared by Raymond andcoworkers for plutonium decorporation (Weitl, et al., J Am Chem Soc,1980, 102 (7): 2289-2293). This octadentate ligand leverages grafting ofcatecholamide (CAM) moieties found in microbial siderophores on thespermine scaffold to form a hybrid version of Ent and 3,4,3-LI(1,2-HOPO)that should (i) display increased complex stability over Ent due to itshigher denticity and (ii) bear more negative charges than3,4,3-LI(1,2-HOPO) due to CAM units requiring further deprotonation formetal binding (FIG. 1). 3,4,3-LI(CAM) was synthesized from readilyavailable building blocks using a process developed in-house (FIG. 10).The new preparation moves away from using harsh reaction conditions byusing the protected diphenylmethylene acetal derivative (5), whichgreatly simplifies purification of the final product.

Affinity of 3,4,3-LI(CAM) Toward 3+ and 4+ Metals. A comprehensivesolution thermodynamic analysis was performed to characterize theaffinity of 3,4,3-LI(CAM) for trivalent and tetravalent metals and theeffect of substituting 1,2-HOPO for CAM binding units on the octadentatespermine scaffold. The protonation constants of 3,4,3-LI(CAM) weredetermined by spectrophotometric titrations, and eight protonationequilibria were assigned to sequential removal of two protons from eachof the four CAM units (FIG. 11). Previous studies of Ent and otherCAM-containing synthetic analogs established that the protonationconstants (pK_(a1)-pK_(a4)) of the meta-hydroxyl oxygen atoms are wellseparated from the ortho-hydroxyl oxygen atoms (pK_(a5)-pK_(a8)) (Loomis& Raymond, Inorganic Chemistry, 1991, 30 (5): 906-911). The last fourpK_(a) values are most relevant to metal binding as moietiescorresponding to these values have to be deprotonated at physiologicalpH in order to bind the metal ions. The overall acidity of 3,4,3-LI(CAM)can be defined as ΣpK_(a5-8)=45.4 versus 3,4,3-LI(1,2-HOPO)'s 21.2(Abergel, et al., Inorganic chemistry 2009, 48 (23): 10868-10870) withlower values representing higher acidity. 3,4,3-LI(CAM) is thereforeless prone to bind hard Lewis acids at low pH than its 1,2-HOPO analog,due to competition between metal uptake and protonation of the CAMmoieties.

Incremental spectrophotometric titrations were then carried out todetermine the formation of Eu^(III), Zr^(IV) or Th^(IV) complexes with3,4,3-LI(CAM). Because of the very short half-life of ²²⁵Ac and thescarce availability of the longer-lived ²²⁷Ac, Eu^(III) was used here asa non-radioactive Ln surrogate for Ac^(III). Based on previous solutionthermodynamic studies of Ln^(III) complexes of 3,4,3-LI(1,2-HOPO) andother common polyaminocarboxylate chelators, 15 it is reasonable toexpect similar stability constants for Eu^(III) and Ac^(III) complexesof 3,4,3-LI(CAM). The CAM octadentate chelator showed a very highaffinity for both 3+ and 4+ ions (FIG. 11). The stability constants of[Eu-3,4,3-LI(CAM)]⁵⁻, [Th-3,4,3-LI(CAM)]⁴⁻ and [Zr-3,4,3-LI(CAM)]⁴⁻ areseveral orders of magnitude higher than those of their 1,2-HOPOcounterparts, with log #β110 values of 29.7, 47.7 and 57.3,respectively. Consequently, 3,4,3-LI(CAM) is one of the strongestchelators ever reported for the chelation of both trivalent andtetravalent f-elements. For comparison, a cyclic octadentateterephthalamide derivative was recently designed to bind Th4+ in vivoand showed an unprecedented affinity for Th4+ with a log #β110 (ThL4−)value of 53.7 (Pham, et al., J. Am. Chem. Soc., 2014, 136 (25):9106-9115). To inspect the pH dependency of metal complex formation,speciation diagrams were calculated for 3,4,3-LI(CAM) in the presence of1 equivalent of Eu(III), Zr(IV) or Th(IV) (FIGS. 5-7). Both Zr(IV) andTh(IV) complexes start forming at around pH 3, with the mono and fullydeprotonated species, [MIVLH]³⁻ and [MIVL]⁴⁻, being predominant atphysiological pH (7.4). This behavior departs from that of3,4,3-LI(1,2-HOPO), with which 4+ metal complexes are formed even undervery acidic conditions (pH<0) (Deblonde, et al., Inorganic chemistry,2013, 52 (15); 8805-8811; Sturzbecher-Hoehne, et al., Inorganicchemistry, 2015, 54 (7): 3462-3468). For Eu(III), complexation by3,4,3-LI(CAM) starts at pH 5 and the mono-protonated complex,[Eu^(III)LH]⁴⁻, is the only species present at pH 7.4. Similar to whatis observed with 4+metals, the pH at which Eu(III)⁻³,4,3-LI(CAM)complexes start forming is higher than in the case ofEu(III)⁻³,4,3-LI(1,2-HOPO) species that already appear at pH 1 underthose same conditions (Abergel, et al., Inorganic chemistry, 2009, 48(23): 10868-10870). However, it is important to note the multiplenegative charges of the 3,4,3-LI(CAM) complexes with 3+ and 4+ metalscomplexes under physiologically relevant conditions ([M^(III)LH]⁴⁻,[M^(IV)LH]³⁻ and [M^(IV)L]⁴⁻), which are now capable of formingelectrostatic interactions with the Scn protein. This represents a largeadvantage over 3,4,3-LI(1,2-HOPO), for which the complexes with M^(III)at pH 7.4 have only one negative charge and are neutral in the case ofM^(IV) ions.

Scn Recognition of 3,4,3-LI(CAM)-Metal Complexes. As described inseveral previous reports (Allred, et al., PNAS, 2015, 112 (33):10342-10347; Abergel, et al., J Am Chem Soc, 2006, 128 (34):10998-10999; Abergel, et al., PNAS 2006, 103 (49): 18499-18503), theaffinity of Scn for chelators or metal-chelator complexes is quantifiedby monitoring protein fluorescence quenching upon ligand or complexbinding. The equilibrium dissociation constant of Scn for the apo formof the chelator 3,4,3-LI(CAM), K_(d)=1.2±0.4 nM, is nearly identical tothat determined for the native siderophore apo-Ent, 11a indicating thatthe addition of a fourth CAM unit does not affect chelator recognitionby the protein. Subsequent determination of K_(d) values for variousmetal complexes of 3,4,3-LI(CAM) (M^(III)=Sm, Eu, Gd, ²⁴³Am, or ²⁴⁸Cm,and M^(IV)=Zr, ²³²Th, or ²⁴²Pu) confirm tight binding to the protein,independent of the metal valence, with values well below 40 nM (FIG.12). As hypothesized, these data demonstrate a large difference inprotein recognition between the 3,4,3-LI(CAM) and 3,4,3-LI(1,2-HOPO)complexes of tetravalent metals. Use of the biprotic CAM units in lieuof the monoprotic 1,2-HOPO moieties led to the formation of negativelycharged complexes, enabling electrostatic interactions with the proteintrilobal calyx. In addition, while the addition of a fourth CAMmetal-binding group in the octadentate 3,4,3-LI(CAM) was important forincreased stability of the metal-ligand complexes at pH 7.4, it did notprevent the high Scn affinities initially observed with hexa-coordinatedEnt complexes. Interestingly, some subtle differences were observed withthe recognition patterns: for similarly charged complexes, weakerbinding was observed with actinide complexes (Am^(III), Cm^(III),Th^(IV), and Pu^(IV)) as compared to corresponding lanthanide or d-blockmetal complexes (Sm^(III), Eu^(III), Gd^(III), Zr^(IV)) in the case of3,4,3-LI(CAM). The opposite trend had been noted with Ent complexes ofM^(III) metals9 and was confirmed here with Zr^(IV), while nosignificant differences are discernable with 3,4,3-LI(1,2-HOPO).

Structural Characterization of Scn-CAM adducts. X-ray crystallographywas used as previously described9 to determine the structures of the Scnadducts formed with the ²³²Th-3,4,3-LI(CAM) and Zr-3,4,3-LI(CAM)complexes (FIG. 9). As expected, and as observed in previous Scn complexstructures (eg., with ²⁴³Am-3,4,3-LI(1,2-HOPO)) (Allred, et al., PNAS,2015, 112 (33): 10342-10347), the compounds bound in the deeply-recessedtrilobal binding site, or calyx, of Scn (FIGS. 13A, 13B, and 13C). As inprevious structures of Scn bound to CAM-bearing ligands with An ions,only one CAM substituent is ordered in the crystal structures along withthe bound metal, Zr or Th. This was likely the result of the remainderof the chelator sampling multiple conformations between molecules in thecrystal, but clearly confirmed binding of chelator and metal in bothadducts. The CAM substituent bound in the key binding pocket in the Scncalyx, between the side-chains of two bracketing lysine residues (K125and K134; FIG. 3D). The structure of the Scn calyx is highly conservedwith prior structures, reflecting its rigidity, with the side-chains oftwo residues, W79 and R81, the only elements flexing to accommodatedifferent chelators (FIG. 13E). In prior Ent or Ent analogchelator/actinide structures (Allred, et al., PNAS, 2015, 112 (33):10342-10347), where two of the three CAM groups are also disordered, theside-chain of W79 adopted an unusual rotamer, flipping inwards towardsthe metal to contribute a cation-π interaction. However, in thesestructures, this side-chain adopts more conventional orientations,either sampling multiple rotamers, rendering it disordered in thecrystallographic analysis (in the Zr complex), or lying against thecalyx wall in the Th complex. Apart from these two side-chains, theoverall impression conveyed by these and previous results is that thechelator flexes and distorts to fit in an essentially rigid andunyielding calyx.

Biodistribution Evaluation. To evaluate the in vivo retention andexcretion patterns of M(IV)-3,4,3-LI(CAM) complexes and their Scnadducts, ²³⁸Pu(IV) was used as a radiolabel. ²³⁸Pu(IV) likely behavessimilarly to Th(IV) and Zr(IV) (the ionic radius of Pu⁴⁺ is includedbetween those of Th⁴⁺ and Zr⁴⁺) (Sturzbecher-Hoehne, et al., InorganicChemistry, 2015, 54 (7): 3462-3468) but allows for more accuratemetabolic balance experiments due to its relatively long radioactivehalf-life (87.8 yr) and low specific activity (0.63 TBq/g), compared tothe therapeutic ²²⁷Th (18.68 d; 1139 TBq/g) and imaging ⁸⁹Zr (78.42 h;16,630 TBq/g) isotopes. It is also important to note that other commonlyavailable isotopes such as ²³²Th (14 Gyr; 4.07 kBq/g) would not exhibitenough activity to allow for radioanalysis. In this in vivo stabilityexperiment, ²³⁸Pu-ligand complex solutions were formed in situ(Ligand:Pu and Scn:Chelator:Pu ratios of 100:1 and 100:100:1,respectively) and administered intravenously. Mice were euthanized 4,24, or 48 h after the metal injection, and tissues and excreta wereradioanalyzed for ²³⁸Pu content (FIG. 14).

Independent of the presence of Scn in the administered solution, 30% ofthe injected ²³⁸Pu was excreted by 48 hours and ²³⁸Pu excreta contentsteadily increased, suggesting delayed clearance of the complexes. Therate of ²³⁸Pu elimination observed for ²³⁸Pu-3,4,3-LI(CAM) is strikinglydifferent from that observed for the ²³⁸Pu-3,4,3-LI(1,2-HOPO) complex inprevious studies (albeit performed in a different strain of mice andwith younger animals), in which quantitative excretion was observed by24 h. In both Scn-bound and free ²³⁸Pu-3,4,3-LI(CAM) cases, and at alltime points, more ²³⁸Pu was found in the urine than in the feces.However, the kidney and liver contents suggest a dramatic difference inexcretion pattern: when free, the 3,4,3-LI(CAM) complex is predominantlyfound in the liver at early time points after administration and followsa biliary pathway, similar to what is known for HOPO complexes. However,insertion within the protein favors elimination through the renalsystem, with up to 52% of ²³⁸Pu found in the kidneys 4 h afteradministration of the Scn adducts, a burden that subsequently slowlydecreases. Combined with significantly faster rates of excretion andconsiderably lower skeleton and soft tissue burden when compared withfree ²³⁸Pu, this major difference between kidney vs. liver ²³⁸Puretention of the Scn-bound vs. free complex evidences the high in vivostability of the Scn:[Pu^(IV)(3,43-LI(CAM))] adduct.

Conclusion. The Scn:3,4,3-LI(CAM) system is a novel and highly promisingchelator platform to develop new radiopharmaceuticals and imagingagents. Scn's highly specific binding to 3,4,3-LI(CAM)-M^(IV) and3,4,3-LI(CAM)-M^(III) complexes eliminates the need for costlybioconjugation of chelators to targeting ligands as the protein may beencoded via well-established biochemical methods. Interestingly, one canenvision a system where both imaging (⁸⁹Zr^(IV)) and therapeutic(²²⁷Th^(IV) or ²²⁵Ac^(III)) metallic radioisotopes may be used inconjunction for dual diagnostics/treatment applications. The describedresults illustrate the promise of this system.

Example 2: Tests with cut-off filters. The present example presents aseparation process that uses size exclusion. Various systems are alreadycommercially available for the purification of macromolecules (includingproteins) from low-molecular weight molecules by size exclusion. Thesesystems usually contain a porous membrane that lets the small moleculespass but retains the macromolecules.

“Cut-off” filters were used for the separation of tin ions (Sn⁴⁺) fromeuropium ions (Eu³⁺). The aim of the tests described in this section wasto obtain an experimental proof of principle of our separation processrather than determining the maximum efficacy or selectivity of theprocess.

Samples containing tin ions complexed to the composition (e.g., ligand)[3,4,3-LI(1,2-HOPO)]⁴⁻ were passed through 3 kDa cut-off filters (2filters from 2 different suppliers). As seen on FIG. 18, the majority ofthe tin ions passed through the filters. The same experiment wasperformed with samples containing europium ions (Eu³⁺) complexed to thecomposition (e.g., ligand) [3,4,3-LI(1,2-HOPO)]⁴⁻ and siderocalin. Inthis case, since a high molecular weight adduct is formed(Scn[Eu(III)L]), the majority of europium is retained by the filters.

The performances obtained above are limited by the performance of thefilters which are not designed for such applications. The fact that lessthan 100% of the tin ions passed through the filters is probably due toadsorption on the membrane. Nonetheless, the results displayed in FIG.18 show that a good separation can be obtained between M⁴⁺ and M³⁺ ionsand even for a 1-step process, which works at room temperature and undermild conditions (aqueous solution at pH=7.4). The separation factorSn/Eu obtained is 41.4 in the case of the GE HealthCare filter and 6.9for the “NanoSep’ filter (FIG. 18).

The influence of the protein itself on the filtration was clarified bypreparing 2 samples: one containing only the complex[Eu(III)-3,4,3-LI(1,2-HOPO)]⁻ and another containing the complex[Eu(III)-3,4,3-LI(1,2-HOPO)]⁻ in addition to siderocalin. FIG. 19 showsthat the addition of the protein allows to form a high-molecular weightadduct that is retained by the filter. Indeed, in the absence of theprotein, 89% of the europium passed through the filter compared to only7.7% under similar conditions but in the presence of the protein.

Example 3: Tests with size exclusion column. An additional sizeexclusion system was tested. A chromatographic size-exclusion media,called “Sephadex G-25”, was used to separate the low-molecular weightcomplex from the high-molecular weight adduct metal-composition (e.g.,ligand)-protein. The Sephadex media is a classical porous size-exclusionmedia used in biology for protein purification. Macromolecules such asproteins are too big to go inside the pores of the media and areconsequently not retained, whereas the small molecules can go inside thepores of the media and their elution is delayed compared to the protein.A scheme is give in FIG. 20. An advantage of such a system, besidesbeing simple and robust, is that the recovery yields can reach 100%, aslong as the column is flushed with the appropriate solution. Once theprotein adduct is eluted, the other components of the initial samples(for example [M(IV)-3,4,3-LI(1,2-HOPO)] complexes) can therefore berecovered by passing more buffer to the column.

Samples containing metal ions and the composition (e.g., ligand)[3,4,3-LI(1,2-HOPO)]⁴⁻ in the presence or in the absence of siderocalinwere injected in a gravity column at room temperature and ambientpressure. The samples were eluted at pH 7.4 with a classical TBS bufferand fractions were collected and analyzed. The first test was performedwith europium ions (Eu³⁺) in order to evaluate the applicability of thissystem. The [Eu(III)-3,4,3-LI(1,2-HOPO)]⁻ complex and thesiderocalin-[Eu(III)-3,4,3-LI(1,2-HOPO)] adduct are fluorescent under UVirradiation and can therefore be followed easily. FIG. 21 illustratesthe separation of the low-molecular weight complex[Eu(III)-3,4,3-LI(1,2-HOPO)]⁻ from thesiderocalin-[Eu(III)-3,4,3-LI(1,2-HOPO)] adduct. The adduct exits thecolumn in the early fractions (5 and 6) whereas the small complex exitsthe column later in fractions 10 to 17.

A quantitative analysis of the fractions depicted above was performed byspectrofluorimetry (FIG. 22Error! Reference source not found.). Theresults given on FIG. 22 confirm the previous qualitative observation(FIG. 21) and show that the macromolecular adduct exits the columnbefore the low molecular complex. These results therefore confirm thatthe presence of the protein clearly influence the elution time of thenegatively charged complexes as exemplified here with[Eu(III)-3,4,3-LI(1,2-HOPO)]⁻.

The size-exclusion column was also tested with the tin(IV) neutralcomplex [Sn(IV)-3,4,3-LI(1,2-HOPO)]. The elution of the complex wasfollowed by UV-vis since the latter is neither luminescent norradioactive but has a characteristic maximum absorbance at 304 nm. FIG.23 shows that the elution of the tin(IV) complex is not recognized bythe protein and that its elution is delayed compared to themacromolecular adducts (comparison FIG. 22 and FIG. 23). Based on theresults obtained with Eu(III) and Sn(IV) it is clear that both metalscan be separated since the Eu(III) complex is recognized by the proteinand exits the column in the early fractions whereas the tin(IV) neutralcomplex is not recognized and elutes long after the macromolecularadduct.

The system was then tested with plutonium ions (Pu⁴⁺). A samplecontaining ²³⁸Pu⁴⁺ ions, the composition (e.g., ligand)[3,4,3-LI(1,2-HOPO)]⁴⁻ and siderocalin was eluted through a Sephadexcolumn and followed by liquid scintillation since the isotope ²³⁸Pu hasa high specific activity (17.1 Ci·g⁻¹). As for[Sn(IV)-3,4,3-LI(1,2-HOPO)], the plutonium neutral complex,[Pu(IV)-3,4,3-LI(1,2-HOPO)], is not recognized by siderocalin and eluteslong after the Scn[Eu(III)L]macromolecular species (comparison of FIG.24 and triangle curve on FIG. 22). It is clear from the results obtainedhere that Eu(III) ions can be separated from both Sn(IV) and Pu(IV) ionsvia by [3,4,3-LI(1,2-HOPO)]/siderocalin-based systems. The sameconclusion can be drawn for the other rare earth ions.

Example 4. A separation experiment was performed for a sample containingboth curium ions (²⁴⁸Cm³⁺) and plutonium ions (²³⁸Pu⁴⁺). Curium andplutonium separation is especially important in the frame of nuclearfuel cycles because these two actinides elements are present in thenuclear wastes and are difficult to separate. The plutonium-curium caseis a good model for the separation of Bk⁴⁺ ions from Cf³⁺ and Cm³⁺. Theplutonium-curium separation is also a good model for the separation ofactinium (Ac³⁺) from thorium (Th⁴⁺) which are two other actinides ofinterest in the context of medical applications.

The presence of plutonium in the different fractions was detected byliquid scintillation. Due to the low activity of the curium isotope used(²⁴⁸Cm, specific activity of 4.3 mCi·g⁻¹), the presence of curiumcouldn't be detected by liquid scintillation but was rather performed byspectrofluorimetry. Indeed, as for Eu(III), the[Cm(III)-3,4,3-LI(1,2-HOPO)]⁻ complex and thesiderocalin-Cm(III)-3,4,3-LI(1,2-HOPO)] adduct are fluorescent under UVirradiation. As expected, the complex formed with [3,4,3-LI(1,2-HOPO)]⁴⁻and Cm³⁺ is recognized by siderocalin which yields a macro-species whichelutes rapidly. On the contrary, the neutral [Pu(IV)-3,4,3-LI(1,2-HOPO)]complex is not up taken by siderocalin and stays longer in the column.It has to be underlined that the initial sample described on FIG. 25 hadhigh ratio curium/plutonium with a value of 25 mol/mol. This ratio isclearly unfavorable to obtain high purity plutonium with classicalseparation processes. Nonetheless, as shown on FIG. 25, the twoactinides elements species have readily different retention times andplutonium fractions without curium could be obtained.

Interestingly, the curium(III) retention time is similar to theeuropium(III) one (comparison of FIG. 25 and FIG. 22) whereas theplutonium(IV) retention time is similar to the tin(IV) one (comparisonof FIG. 25 and FIG. 23). These results show that the separation doesn'tdepend on the nature of the metal ion but on the charge of its complexwith the small composition (e.g., ligand) (here [3,4,3-LI(1,2-HOPO)]⁴⁻).

The results described above demonstrate that ions can be separated in anefficient manner by using protein-composition (e.g., ligand) recognitionand subsequent selective formation of high-molecular weight species. Theproofs of concept given above pave the way for new separation orpurification processes. All the separations described in these isolationexperiments were performed at room temperature, at ambient pressure,using a one-step process and under mild chemical conditions (fullyaqueous solvent, pH 7.4). Moreover, the chemical or biochemical reagentsemployed do not contain non-volatile elements which could allow thefinal recovery and concentration of the purified ion by simpletechniques such as ignition. The system described above also exhibits ahigh selectivity and allows separating and purifying ions even forsamples that present a very unfavorable metal ions ratio (asdemonstrated in the Cm³⁺/Pu⁴⁺ experiment). The results also indicatethat the system selectivity is generic for ions having the sameelectronic charge (for example Cm³⁺ and Eu³⁺ versus Sn⁴⁺ and Pu⁴⁺) whichopen ways to several applications.

Example 5. The oxidation state of Bk when bound to 3,4,3-L(1,2-HOPO) wasunambiguously assigned through liquid chromatography (LC) coupled withhigh resolution mass spectrometry (MS). Analysis of 1:1 metal:ligandaqueous mixtures prepared under ambient conditions with ²⁴¹Am, ²⁴⁸Cm and²⁴⁹Cf, whose M⁴⁺/M³⁺ redox potentials are extremely high ([Am], +3.1 and+3.2 V, respectively), confirmed the formation of trivalent3,4,3-LI(1,2-HOPO) complexes. For those three trans-Pu elements, the MSpatterns are almost identical, with four mono-charged adducts detected([M^(III)LH₂]⁺, [M^(III)LHNa]⁺, [M^(III)LNa₂]⁺ and [M^(III)LNaK]⁺),which clearly contrasts with the data obtained for tetravalent ²⁴²Pu and²³²Th complexes. The MS spectrum of the ²⁴⁹Bk system assembled in situfrom a BkCl₃ solution displayed [BkLH]⁺, [BkLNa]⁺ and [BkLK]⁺ species,evidently demonstrating that the Bk complex contains a Bk(IV) ion andnot Bk(III). Spontaneous oxidation of Bk(III) to Bk(IV) is thought tooccur through air oxidation, similarly to the Ce system, which does notnecessitate the addition of oxidizers or electrolytic oxidation requiredin previously proposed methods. The use of 3,4,3-LI(1,2-HOPO) as achelation and oxidation-promoting agent for Bk also has the notableadvantage of promoting the formation of M(IV) complexes over a widepH-range: the Zr(IV), Ce(IV) and Pu(IV) complexes are formed in 1 MH₂SO₄ and are stable up to pH 11.

Liquid Chromatography-Mass Spectrometry. The experimental setting usedfor liquid chromatography-high resolution mass spectrometry assays(LC-HRMS) has been previously described (M. Sturzbecher-Hoehne, T. A.Choi, R. J. Abergel, Hydroxypyridinonate Complex Stability of Group (IV)Metals and Tetravalent f-Block Elements: The Key to the Next Generationof Chelating Agents for Radiopharmaceuticals, Inorg. Chem. 54 (2015)3462-3468. doi:10.1021/acs.inorgchem.5b00033). LC-HRMS spectra wereacquired on a UPLC Waters Xevo system interfaced with a QTOF massspectrometer (Waters Corporation, Milford, Mass., USA) in MicromassZ-spray geometry. Chromatographic separation was achieved on ananalytical Zorbax Eclipse column (Agilent Technologies, XDB-C18, 5 μm,4.6×150 mm) maintained at ambient temperature (25° C.) with two mobilephases (water (A) and methanol (B)) containing 0.5% formic acid. Samples(10 μL injection) were eluted using a gradient initially held constantat 7% B for 6.0 min and were then progressed to 40% B in the next 6.0min and held at 40% B for 10 min. Mobile phase B was then increased to99% over 3.0 min, held constant at 99% for 5.0 min, and then rapidlyswitched to 7% B and held until 46 min for equilibration. The flow ratewas maintained at 0.5 mL/min. The mass spectrometer equipped with an ESIsource was operated in positive ion mode, and mass spectra were acquiredin the continuum mode across the m/z range of 100-1200, at 5 s per scan,with a 14 ms interscan delay. Data acquisition and instrument controlwere accomplished using MassLynx software, version 4.1. Samples wereinfused into the ionization chamber from the LC system. The operatingparameters were as follows: the nebulization gas flow rate was set to600 L/h with a desolvation temperature of 375° C., the cone gas flowrate was set to 30 L/h, and the ion source temperature was 125° C. Thecapillary, sampling cone, and extraction cone voltages were tuned to 2.7kV, 47 V, and 3.3 V, respectively. Liquid nitrogen served as nebulizerand argon was used as collision gas with collision energies up to 50 eV.A calibration check of the instrument was performed with 0.5 mM sodiumformate, prior to sample analysis. Samples containing an equalconcentration of actinide and 3,4,3-LI(1,2-HOPO) were prepared in 0.1 MHEPES buffer at pH 7.4 (for Cm, Cf and Bk) or in 0.5% formic acid at pH2 (for Ce, Th, and Pu). The concentrations used were 10 μM for ²⁴³Am,²⁴⁹Bk and ²⁴⁹Cf samples and 1 μM for Ce, ²³²Th, ²⁴²Pu, and ²⁴⁸Cm. Forconsistency, an addition of 0.1 μM of [Zr^(IV)3,4,3-LI(1,2-HOPO)] wasperformed in each sample in order to use the Zr complex as internalreference. The retention times of independent samples were thennormalized using that of [Zr^(IV)3,4,3-LI(1,2-HOPO)].

As will be understood by one of ordinary skill in the art, eachembodiment disclosed herein can comprise, consist essentially of, orconsist of its particular stated element, step, ingredient or component.Thus, the terms “include” or “including” should be interpreted torecite: “comprise, consist of, or consist essentially of.” Thetransition term “comprise” or “comprises” means includes, but is notlimited to, and allows for the inclusion of unspecified elements, steps,ingredients, or components, even in major amounts. The transitionalphrase “consisting of” excludes any element, step, ingredient orcomponent not specified. The transition phrase “consisting essentiallyof” limits the scope of the embodiment to the specified elements, steps,ingredients or components and to those that do not materially affect theembodiment. A material effect would cause a statistically significantreduction in the ability of Scn, chelators, and radionuclides to formSCC complexes, and/or in the ability of a particular SCC complex toprovide a therapeutically effective treatment according to an objectivemeasure disclosed herein.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that can varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques. When further clarity is required, the term “about” has themeaning reasonably ascribed to it by a person skilled in the art whenused in conjunction with a stated numerical value or range, i.e.denoting somewhat more or somewhat less than the stated value or range,to within a range of ±20% of the stated value; ±19% of the stated value;±18% of the stated value; ±17% of the stated value; ±16% of the statedvalue; ±15% of the stated value; ±14% of the stated value; ±13% of thestated value; ±12% of the stated value; ±11% of the stated value; ±10%of the stated value; ±9% of the stated value; ±8% of the stated value;±7% of the stated value; ±6% of the stated value; ±5% of the statedvalue; ±4% of the stated value; ±3% of the stated value; ±2% of thestated value; or +1% of the stated value.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements.

The terms “a,” “an,” “the”, and similar referents used in the context ofdescribing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group can be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents, printedpublications, journal articles and other written text throughout thisspecification (referenced materials herein). Each of the referencedmaterials is individually incorporated herein by reference in theirentirety for their referenced teaching.

The particulars shown herein are by way of example and for purposes ofillustrative discussion of the preferred embodiments of the presentinvention only and are presented in the cause of providing what isbelieved to be the most useful and readily understood description of theprinciples and conceptual aspects of various embodiments of theinvention. In this regard, no attempt is made to show structural detailsof the invention in more detail than is necessary for the fundamentalunderstanding of the invention, the description taken with the drawingsand/or examples making apparent to those skilled in the art how theseveral forms of the invention can be embodied in practice.

Definitions and explanations used in the present disclosure are meantand intended to be controlling in any future construction unless clearlyand unambiguously modified in the following examples or when applicationof the meaning renders any construction meaningless or essentiallymeaningless. In cases where the construction of the term would render itmeaningless or essentially meaningless, the definition should be takenfrom Webster's Dictionary, 3rd Edition or a dictionary known to those ofordinary skill in the art, such as the Oxford Dictionary of Biochemistryand Molecular Biology (Ed. Anthony Smith, Oxford University Press,Oxford, 2004).

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that can be employed are within the scopeof the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention can be utilized inaccordance with the teachings herein. Accordingly, the present inventionis not limited to that precisely as shown and described.

What is claimed is:
 1. A composition comprising a compound or a saltthereof bound to a metal and a siderocalin, the compound having thestructure of Formula I:

wherein: (i) each of A1, A2, A3, and A4, individually, is a CAM group, a1,2-HOPO group, or a HA group; (ii) each of B1, B2, B3, and B4,individually, is an amide group or an amine group; (iii) at least one ofC1, C2, C3, C4, C5, and C6, individually, is SH; (iv) at least anotherone of C1, C2, C3, C4, C5, and C6 is optional and, individually, isC(═O)OH or NH₂; (v) at least one of L1, L2, L3, L4, L5, L6, L7, L8, L9,L10, L11, L12, and L13, individually, is H, amine group, amide group, analkyl group having no greater than 10 carbon atoms, an alkylamino grouphaving no greater than 10 carbon atoms and no greater than 2 nitrogenatoms; an alkyl ether group having no greater than 10 carbon atoms, ahydroxy ester group, or an alkyl ester group having no greater than 10carbon atoms; and (vi) at least one of L1, L5, L6, L7, L8, L9, L10, L11,L12, and L13 is optional.
 2. The composition of claim 1, the compoundhaving the structure of Formula I, wherein: at least another one of L2,L3, and L4, individually, is an amine group or an amide group; or whenL1, C1, L7, C2, L9, C3, L11, C4, L13, and C5 are absent, L5 is anunsubstituted alkyl group having no greater than 5 carbon atoms, and C6is SH.
 3. The composition of claim 1, the compound having the structureof Formula I, wherein each of L2, L3, L4, L6, L8, L10, and L12,individually, is an unsubstituted alkyl group having no greater than 5carbon atoms; and optionally wherein: A1 is a CAM group or a HOPO group;A2 is a HA group; A3 is a HA group; and A4 is a CAM group, a HOPO group,or a HA group.
 4. The composition of claim 1, the compound having thestructure of Formula I, wherein: (i) at least one of L2, L3, or L4,individually, is an alkylamino group; or (ii) each of B1, B2, and B3,individually, is an amide group; B4 is an amino group; each of L2 andL3, individually, is an amino group; and L4 is an alkyl group having nogreater than 5 carbon atoms; and optionally, when C1, C2, C3, C4, C5,A1, A2, A3, L1, L6, L7, L8, L9, L10, L11, L12, and L13 are absent, A4 isa CAM group, a HOPO group, or a HA group; and L5 is an alkyl grouphaving no greater than 5 carbon atoms.
 5. The composition of claim 1,the compound having the structure of Formula I, wherein: (i) each of B1,B2, and B3, individually, is an amide group; B4 is an amino group; eachof L2 or L3, individually, is an amino group; and L4 is an alkyl grouphaving no greater than 5 carbon atoms; and optionally, when C1, C2, C3,C4, C5, A1, A2, A3, L1, L6, L7, L8, L9, L10, L11, and L13 are absent,L12 is an amino group, L5 is an ether group having no greater than 10carbon atoms, and A4 is a CAM group, a HOPO group, or a HA group; or(ii) when C1, C2, C5, C6, L1, L2, L3, L4, L5, L7, L13, B2, and B4 areabsent, each of 1 and B3, individually, is an amide group; each of L6,L8, L10, and L12, individually, is an amino group; each of A1, A2, A3,and A4, individually, is a CAM group, a HOPO group, or a HA group; andeach of L9 and L11, individually, is an alkyl group having no greaterthan 5 carbon atoms.
 6. A composition comprising a compound or a saltthereof bound to a metal and a siderocalin, the compound having thestructure of Formula II:

wherein: at least one of R₁, R₂, R₃, R₄, and R₅, individually, is a CAMgroup, a HA group, or a 1,2-HOPO group; at least another one of R₁, R₂,R₃, R₄, and R₅, individually, is H, an alkyl group having from 1 to 10carbon atoms; R₆ is (i) H, (ii) an alkyl group having from 1 to 10carbon atoms, or (iii) an alkyl group having from 1 to 10 carbon atomsand substituted by at least one of SH, NH₂, or C(═O)OH; m can be from 1to 6; n can be from 1 to 6; and o can be from 1 to
 6. 7. The compositionof claim 6, the compound having the structure of Formula III:

wherein: at least one of R₁, R₂, R₃, R₄, and R₅, individually, is a CAMgroup, a HA group, or a 1,2-HOPO group; optionally, another one of R₁,R₃, R₄, or R₅, individually, is H or an alkyl group having from 1 to 10carbon atoms; R₂ is H or an alkyl group including from 1 to 5 carbonatoms; R₇ is SH, C(═O)OH, or NH₂; and p is from 1 to 4; and optionally,wherein: R₁ is a CAM group or a 1,2-HOPO group; each of R₃ and R₄,individually, is a HA group; and R₅ is a CAM group, a 1,2-HOPO group, ora HA group.
 8. The composition of claim 7, the compound having thestructure of Formula IV, V, VI or VII:

wherein: R₇ is SH, NH₂, or C(═O)OH; each of R₂, R₈, and R₉,individually, is H or an alkyl group including from 1 to 5 carbon atoms;and p is from 1 to
 4. 9. The composition of claim 7, the compound havingthe structure of Formula VIII or IX:

wherein: R₇ is SH, C(═O)OH, or NH₂; each of R₂, R₈, R₉, and R₁₀,individually, is an H or an alkyl group including from 1 to 5 carbonatoms; and p is from 1 to
 4. 10. The composition of claim 7, thecompound having the structure of Formula X or XI:


11. A composition comprising a compound or a salt thereof bound to ametal and a siderocalin, the compound having the structure of FormulaXII:

wherein: at least one of R₁₁, R₁₂, R₁₃, or R₁₅, individually, is a CAMgroup, a HA group, or a 1,2-HOPO group; optionally, at least another oneof R₁₁, R₁₂, R₁₃, or R₁₅, individually, is H, OH, or an alkyl grouphaving from 1 to 10 carbon atoms; R₁₇ is SH, C(═O)OH, or NH₂; each ofR₂, R₁₄, and R₁₆, individually, is H, OH, or an alkyl group having from1 to 10 carbon atoms; and r can be from 0 to 6; and optionally, whereinR₁₁ is a CAM group or a 1,2-HOPO group; R₁₂ and R₁₅, individually, is aHA group; and R₁₃ is a CAM group, a 1,2-HOPO group, or a HA group. 12.The composition of claim 11, the compound having the structure ofFormula XIII, XIV, XVII, or XVIII:

wherein: each of R₂, R₁₄, R₁₆, R₁₈, and R₁₉, individually, is H, OH, oran alkyl group having from 1 to 10 carbon atoms; R₁₇ is SH, C(═O)OH, orNH₂; and r can be from 0 to
 4. 13. The composition of claim 11, thecompound having the structure of Formula XV or XVI:

wherein: each of R₂, R₁₄, R₁₆, R₁₃, R₁₉, and R₂₀, individually, is H,OH, or an alkyl group having from 1 to 10 carbon atoms; R₁₇ is SH,C(═O)OH, or NH₂; and r can be from 0 to
 4. 14. A composition comprisinga compound or a salt thereof bound to a metal and a siderocalin, thecompound having the structure of Formula XIX,

wherein: each of R₂₁ and R₂₂, individually, is H, OH, or an alkyl grouphaving from 1 to 10 carbon atoms; R₂₃ is H, OH, an alkyl group havingfrom 1 to 10 carbon atoms, or (CH₂)_(e)R_(a), wherein R_(a) is SH; R₂₄is a substituent that includes a CAM group, a 1,2-HOPO group, or a HAgroup; each of a, b, and c, individually, is from 1 to 10; and d is from1 to 4; and e is from 1 to 10; and optionally wherein R₂₄ includes SH,C(═O)OH, or NH₂.
 15. A composition comprising a compound or a saltthereof bound to a metal and a siderocalin, the compound having thestructure of: i) Formula XX or XXIV:

wherein: each of R₂₅, R₂₆, and R₂₇, individually, is H, OH, or an alkylgroup having from 1 to 10 carbon atoms; R₂₈ is H, an alkyl group having1 to 5 carbon atoms, SH, C(═O)OH, or NH₂; and s is from 0 to 4, ii)Formula XXI or XXII

wherein: each of R₂₅, R₂₆, R₂₇, and R₃₀, individually, is H, OH, or analkyl group having from 1 to 10 carbon atoms; at least one of R₂₈ orR₂₉, individually, is SH and the other of R₂₈ or R₂₉ is H, an alkylgroup having from 1 to 5 carbon atoms, SH, NH₂, or C(═O)OH; s is from 0to 4; and t is from 0 to 4; iii) Formula XXIII:

wherein: each of R₂₅, R₂₆, R₂₇, R₃₀, and R₃₁, individually, is H, OH, oran alkyl group having from 1 to 10 carbon atoms; at least one of R₂₈ orR₂₉, individually, is SH and the other of R₂₈ or R₂₉ is H, an alkylgroup having from 1 to 5 carbon atoms, SH, NH₂, or C(═O)OH; s is from 0to 4; and t is from 0 to 4; or iv) Formula XXV:

wherein: each of R₂₅, R₂₆, R₂₇, and R₃₂, individually, is H, OH, or analkyl group having from 1 to 10 carbon atoms; R₂₈ is H, an alkyl grouphaving 1 to 5 carbon atoms, SH, C(═O)OH, or NH₂; and s is from 0 to 4.16. The composition of claim 1, the compound having the structure ofFormula XXVI:

wherein: each of A, B, C, and D, individually, is one or more amidegroups, one or more amine groups, or an alkyl group having from 1 to 10carbon atoms; each of R₃₃, R₃₄, R₃₅, and R₃₆, individually, is a CAMgroup, a 1,2-HOPO group, or a HA group and at least one of R₃₃, R₃₄,R₃₅, or R₃₆ is substituted by SH; and each of g, h, i, and j,individually, is from 1 to
 10. 17. The composition of claim 16, thecompound having the structure of Formula XXVII, XXVIII, or XXIX:

wherein: at least one of R₃₇ or R₄₂, individually, is SH and the otherof R₃₇ or R₄₂ is H, an alkyl group having from 1 to 5 carbon atoms, SH,C(═O)OH, or NH₂; each of R₃₈, R₃₉, R₄₀, and R₄₁, individually, is H, OH,or an alkyl group having from 1 to 5 carbon atoms; and each of u and v,individually, is from 0 to
 5. 18. The composition of claim 1, whereinthe siderocalin comprises any of SEQ ID NOs. 1-25; or wherein thesiderocalin is a mutated siderocalin with position 54 modified fromthreonine to cysteine, position 68 modified from serine to cysteine, orboth position 54 modified from threonine to cysteine and position 68modified from serine to cysteine; and optionally, wherein the mutedsiderocalin is bound to the compound at position 54, position 68, orboth position 54 and position
 68. 19. The composition of claim 1,wherein the metal is a radionuclide; and optionally, wherein theradionuclide comprises ²²⁵Ac, ²²⁶Ac, ²²⁸Ac, ¹⁰⁵Ag, ¹⁰⁶mAg, ¹¹⁰mAg,¹¹¹Ag, ¹¹²Ag, ¹¹³Ag, ²³⁹Am, ²⁴⁰Am, ²⁴²Am, ²⁴⁴Am, ³⁷Ar, ⁷¹As, ⁷²As, ⁷³As,⁷⁴As, ⁷⁶As, ⁷⁷As, ²⁰⁹At, ²¹⁰At, ¹⁹¹Au, ¹⁹²Au, ¹⁹³Au, ¹⁹⁴Au, ¹⁹⁵Au,¹⁹⁶Au, ¹⁹⁶m²Au, ¹⁹⁸Au, ¹⁹⁸mAu, ¹⁹⁹Au, ²⁰⁰mAu, ¹²⁸Ba, ¹³¹Ba, ¹³³mBa,¹³⁵mBa, ¹⁴⁰Ba, ⁷Be, ²⁰³Bi, ²⁰⁴Bi, ²⁰⁵Bi, ²⁰⁶Bi, ²¹⁰Bi, ²¹²Bi, ²⁴³Bk,²⁴⁴Bk, ²⁴⁵Bk, ²⁴⁶Bk, ²⁴⁸mBk, ²⁵⁰Bk, ⁷⁶Br, ⁷⁷Br, ⁸⁰mBr, ⁸²Br, ¹¹C, ¹⁴C,⁴⁵Ca, ⁴⁷Ca, ¹⁰⁷Cd, ¹¹⁵Cd, ¹¹⁵mCd, ¹¹⁷mCd, ¹³²Ce, ¹³³mCe, ¹³⁴Ce, ¹³⁵Ce,¹³⁷Ce, ¹³⁷mCe, ¹³⁹Ce ¹⁴¹Ce ¹⁴³Ce ¹⁴⁴Ce ²⁴⁶Cf, ²⁴⁷Cf, ²⁵³Cf, ²⁵⁴Cf,²⁴⁰Cm, ²⁴¹Cm, ²⁴²Cm, ²⁵²Cm, ⁵⁵Co, ⁵⁶Co, ⁵⁷Co, ⁵⁸Co, ⁵⁸mCo, ⁶⁰Co, ⁴⁸Cr,⁵¹Cr, ¹²⁷Cs, ¹²⁹Cs, ¹³¹Cs, ¹³²Cs, ¹³⁶Cs, ¹³⁷Cs, ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu,¹⁵³Dy, ¹⁵⁵Dy, ¹⁵⁷Dy, ¹⁵⁹Dy, ¹⁶⁵Dy, ¹⁶⁶Dy, ¹⁶⁰Er, ¹⁶¹Er, ¹⁶⁵Er, ¹⁶⁹Er,¹⁷¹Er, ¹⁷²Er, ²⁵⁰Es, ²⁵¹Es, ²⁵³Es, ²⁵⁴Es, ²⁵⁴mEs, ²⁵⁵Es, ²⁵⁶mEs, ¹⁴⁵Eu,¹⁴⁶Eu, ¹⁴⁷Eu, ¹⁴⁸Eu, ¹⁴⁹Eu, ¹⁵⁰mEu, ¹⁵²mEu, ¹⁵⁶Eu, ¹⁵⁷Eu, ⁵²Fe, ⁵⁹Fe,²⁵¹Fm, ²⁵²Fm, ²⁵³Fm, ²⁵⁴Fm, ²⁵⁵Fm, ²⁵⁷Fm, ⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga, ⁷²Ga, ⁷³Ga,¹⁴⁶Gd, ¹⁴⁷Gd, ¹⁴⁹Gd, ¹⁵¹Gd, ¹⁵³Gd ¹⁵⁹Gd⁶⁸Ge, ⁶⁹Ge, ⁷¹Ge, ⁷⁷Ge, ¹⁷⁰Hf,¹⁷¹Hf, ¹⁷³Hf, ¹⁷⁵Hf, ¹⁷⁹m²Hf, ¹⁸⁰mHf, ¹⁸¹Hf, ¹⁸⁴Hf, ¹⁹²Hg, ¹⁹³Hg,¹⁹³mHg, ¹⁹⁵Hg, ¹⁹⁵mHg, ¹⁹⁷Hg, ¹⁹⁷mHg, ²⁰³Hg, ¹⁶⁰mHo, ¹⁶⁶Ho, ¹⁶⁷Ho, ¹²³I,¹²⁴I, ¹²⁶I, ¹³⁰I, ¹³²I, ¹³³I, ¹³⁵I, ¹⁰⁹In¹¹⁰In, ¹¹¹In, ¹¹⁴m In, ¹¹⁵m In,¹⁸⁴Ir, ¹⁸⁵Ir, ¹⁸⁶Ir, ¹⁸⁷Ir, ¹⁸⁸Ir, ¹⁸⁹Ir, ¹⁹⁰Ir, ¹⁹⁰m²Ir, ¹⁹²Ir, ¹⁹³mIr, ¹⁹⁴Ir, ¹⁹⁴m²Ir, ¹⁹⁵m Ir, ⁴²K, ⁴³K, ⁷⁶Kr, ⁷⁹Kr, ⁸¹mKr, ⁸⁵mKr, ¹³²La,¹³³La, ¹³⁵La, ¹⁴⁰La, ¹⁴¹La, ²⁶²Lr, ¹⁶⁹Lu, ¹⁷⁰Lu, ¹⁷¹Lu, ¹⁷²Lu, ¹⁷⁴mLu,¹⁷⁶mLu, ¹⁷⁷Lu, ¹⁷⁷mLu, ¹⁷⁹Lu, ²⁵⁷Md, ²⁵⁸Md, ²⁶⁰Md, ²⁸Mg, ⁵²Mn, ⁹⁰Mo,⁹³mMo, ⁹⁹Mo, ¹³N, ²⁴Na, ⁹⁰Nb, ⁹¹mNb, ⁹²mNb, ⁹⁵Nb, ⁹⁵mNb, ⁹⁶Nb, ¹³⁸Nd,¹³⁹mNd, ¹⁴⁰Nd, ¹⁴⁷Nd, ⁵⁶Ni, ⁵⁷Ni, ⁶⁶Ni, ²³⁴Np, ²³⁶mNp, ²³⁸Np, ²³⁹Np,¹⁵O, ¹⁸²Os, ¹⁸³Os, ¹⁸³mOs, ¹⁸⁵Os, ¹⁸⁹mOs, ¹⁹¹Os, ¹⁹¹mOs, ¹⁹³Os, ³²P,³³P, ²²⁸Pa, ²²⁹Pa, ²³⁰Pa, ²³²Pa, ²³³Pa, ²³⁴Pa, ²⁰⁰Pb, ²⁰¹Pb, ²⁰²mPb,²⁰³Pb, ²⁰⁹Pb, ²¹²Pb, ¹⁰⁰Pd, ¹⁰¹Pd, ¹⁰³Pd, ¹⁰⁹Pd, ¹¹¹mPd, ¹¹²Pd, ¹⁴³Pm,¹⁴⁸Pm, ¹⁴⁸mPm, ¹⁴⁹Pm, ¹⁵¹Pm, ²⁰⁴Po, ²⁰⁶Po, ²⁰⁷Po, ²¹⁰Po, ¹³⁹Pr, ¹⁴²Pr,¹⁴³Pr, ¹⁴⁵Pr, ¹⁸⁸Pt, ¹⁸⁹Pt, ¹⁹¹Pt, ¹⁹³mPt, ¹⁹⁵mPt, ¹⁹⁷Pt, ²⁰⁰Pt, ²⁰²Pt,²³⁴Pu, ²³⁷Pu, ²⁴³Pu, ²⁴⁵Pu, ²⁴⁶Pu, ²⁴⁷Pu, ²²³Ra, ²²⁴Ra, ²²⁵Ra, ⁸¹Rb,⁸²Rb, ⁸²mRb, ⁸³Rb, ⁸⁴Rb, ⁸⁶Rb, ¹⁸¹Re, ¹⁸²Re, ¹⁸²mRe, ¹⁸³Re, ¹⁸⁴Re,¹⁸⁴mRe, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ¹⁹⁰mRe, ⁹⁹Rh, ⁹⁹mRh, ¹⁰⁰Rh, ¹⁰¹mRh, ¹⁰²Rh,¹⁰³mRh, ¹⁰⁵Rh, ²¹¹Rn, ²²²Rn, ⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru, ³⁵S, ¹¹⁸mSb, ¹¹⁹Sb,¹²⁰Sb, ¹²⁰mSb, ¹²²Sb, ¹²⁴Sb, ¹²⁶Sb, ¹²⁷Sb, ¹²⁸Sb, ¹²⁹Sb, ⁴³Sc, ⁴⁴Sc,⁴⁴mSc, ⁴⁶Sc, ⁴⁷Sc, ⁴⁸Sc, ⁷²Se, ⁷³Se, ⁷⁵Se, ¹⁵³Sm, ¹⁵⁶Sm, ¹¹⁰Sn, ¹¹³Sn,¹¹⁷mSn, ¹¹⁹mSn, ¹²¹Sn, ¹²³Sn, ¹²⁵Sn, ⁸²Sr, ⁸³Sr, ⁸⁵Sr, ⁸⁹Sr, ⁹¹Sr,¹⁷³Ta, ¹⁷⁵Ta, ¹⁷⁶Ta, ¹⁷⁷Ta, ¹⁸⁰Ta, ¹⁸²Ta, ¹⁸³Ta, ¹⁸⁴Ta, ¹⁴⁹Tb, ¹⁵⁰Tb,¹⁵¹Tb, ¹⁵²Tb, ¹⁵³Tb, ¹⁵⁴Tb, ¹⁵⁴mTb, ¹⁵⁴m²Tb, ¹⁵⁵Tb, ¹⁵⁶Tb, ¹⁵⁶mTb,¹⁵⁶m²Tb, ¹⁶⁰Tb, ¹⁶¹Tb, ⁹⁴Tc, ⁹⁵Tc, ⁹⁵mTc, ⁹⁶Tc, ⁹⁷mTc, ⁹⁹mTc, ¹¹⁸Te,¹¹⁹Te, ¹¹⁹mTe, ¹²¹Te, ¹²¹mTe, ¹²³mTe, ¹²⁵mTe, ¹²⁷Te, ¹²⁷mTe, ¹²⁹mTe,¹³¹mTe, ¹³²Te, ²²⁷Th, ²³¹Th, ²³⁴Th, ⁴⁵Ti, ¹⁹⁸Tl, ¹⁹⁹Tl, ²⁰⁰Tl, ²⁰¹Tl,²⁰²Tl, ²⁰⁴Tl, ¹⁶⁵Tm, ¹⁶⁶Tm, ¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁷⁰Tm, ¹⁷²Tm, ¹⁷³Tm, ²³⁰U,²³¹U, ²³⁷U, ²⁴⁰U, ⁴⁸V, ¹⁷⁸W, ¹⁸¹W, ¹⁸⁵W, ¹⁸⁷W, ¹⁸⁸W, ¹²²Xe, ¹²⁵Xe,¹²⁷Xe, ¹²⁹mXe, ¹³¹mXe, ¹³³Xe, ¹³³mXe, ¹³⁵Xe, ⁸⁵mY, ⁸⁶Y, ⁸⁷Y, ⁸⁷mY, ⁸⁸Y,⁹⁰Y, ⁹⁰mY, ⁹¹Y, ⁹²Y, ⁹³Y, ¹⁶⁶Yb, ¹⁶⁹Yb, ¹⁷⁵Yb, ⁶²Zn, ⁶⁵Zn, ⁶⁹mZn, ⁷¹mZn,⁷²Zn, ⁸⁶Zr, ⁸⁸Zr, ⁸⁹Zr, ⁹⁵Zr, and/or ⁹⁷Zr.